# Patent

## Title
Methods for small RNA sequencing

## Abstract

Next generation sequencing technologies are becoming a preferred method for sequencing nucleic acids and profiling miRNAs. Experimental results disclosed herein show that the most common platform for preparing nucleic acids such as miRNAs for sequencing introduces serious biases. Provided herein are compositions and methods for improved sequencing and miRNA profiling using a set of customized ligation adaptors.

## Background
RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Application No. 61/519,023, filed May 13, 2011, the disclosure of which is incorporated by reference herein in its entirety, including drawings.


TABLE SUBMITTED ON COMPACT DISK
Table 2 is contained on a CD-R provided herewith under the file name “54435.8095.US02 Table 2.txt.” This file is 194 kb and was created on May 11, 2012. The machine format is IBM-PC and the operating system compatibility is MS-Windows. Two copies of the CD-R containing Table 2 are provided pursuant to 37 C.F.R. §1.52(e)(iii). The complete contents of the CD-R are hereby incorporated by reference herein. Any reference to Table 2 in the specification represents an incorporation by reference of the contents of the file 54435.8095.US02 Table 2.txt at that particular location in the specification. 







LENGTHY TABLES




The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US08859239B2). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).






SEQUENCE LISTING SUBMITTED ON COMPACT DISK
The sequence listing in computer readable format is contained on a CD-R provided herewith under the file name “Sequence Listing.txt.” This file is 15 kb and was created on May 11, 2012. The machine format is IBM-PC and the operating system compatibility is MS-Windows. Two copies of the CD-R containing the sequence listing in computer readable format are provided pursuant to 37 C.F.R. §1.824. The complete contents of the CD-R are hereby incorporated by reference herein. A paper copy of the sequence listing is also provided.
BACKGROUND
Small RNAs (smRNAs) encompass several different classes of non-coding RNAs, including microRNAs (miRNAs), short interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), and small nuclear RNAs (snRNAs). Among the endogenous smRNAs, miRNA is the most well studied with regard to both biogenesis and functional mechanism. miRNAs are short RNA molecules that act as post-transcriptional regulators by binding to mRNA and preventing it from being translated. The first miRNA, Lin-4, was identified in C. elegans in 1993 (Lee 1993; Wightman 1993). In 2000, a second miRNA, Let-7, was identified and found to be conserved across many species (Pasquinelli 2000; Reinhart 2000). In 2001, it was disclosed that miRNAs probably exist in all species (Lee 2001; Lau 2001; Lagos-Quintana 2001). Since 2001, miRNA research has extended to almost all corners of biological science, with in-depth investigations into miRNA biogenesis and biological functions and the use of miRNA as a therapeutic tool, diagnosis and prognosis marker, and treatment response predictor marker (Galasso 2010; Nagpal 2010; Kim 2009; Bartel 2009). This progression in miRNA study has coincided with the identification and profiling of novel smRNAs in many organisms.
smRNA profiling have traditionally relied on cloning and sequencing of individual RNAs using standard molecular methods. In the most common approach, adaptor oligonucleotides are joined to the 3′ and 5′ termini of smRNAs, and the ligation products are reverse transcribed and PCR amplified to generate a cDNA library. This procedure represents a significant technical challenge because it requires three gel purification steps. In addition, thousands of clones have to be individually sequenced to identify the smRNA population. This standard protocol is labor intensive, painstaking, and requires large amounts of starting materials, and therefore is not practical for many research or clinical settings (Pfeffer 2005). Although cost can be reduced several fold by concatenating fragments of smRNAs with both adaptors to sequence several clones together, expense is still a major obstacle in thoroughly surveying smRNA populations.
Next generation sequencing (NGS) technology was first applied to smRNA discovery with the use of massive parallel signature sequencing to survey the smRNA library of Arabidopsis thaliana (Lu 2005). Since then, many modified smRNA profiling procedures based on NGS have been developed and tested on various platforms (Hafner 2008; Lu 2007; Tang 2010). To date, human miRNAs alone represent 1,048 unique sequence entries in miRBase 16 (Griffiths-Jones 2010). NGS technology has also helped in the discovery of other smRNAs (Lu 2005).
Illumina's NGS technology (Solexa) has been rated one of the two leading protocols for RNA sequencing (Levin 2010). The Solexa platform has gained some advantage over other smRNA profiling protocols with their smRNA cloning protocol v1.5. This protocol, which is summarized in FIG. 1, drastically reduces the difficulties of smRNA cDNA library construction by eliminating the need for gel purification of smRNA/smRNA-adaptors (the most challenging and critical steps in smRNA discovery) and by requiring only about 1 μg of total RNA. However, it has been found that this protocol presents a major artifact in smRNA sequencing experiments. Therefore, there is a need in the art for improved methods of smRNA sequencing.
SUMMARY
Provided herein in certain embodiments are isolated polynucleotides comprising, consisting of, or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs:2 to 65.
Provided herein in certain embodiments are compositions comprising one or more isolated polynucleotides each independently comprising, consisting of, or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs:2 to 65. In certain of these embodiments, the composition comprises a specific set of two or more isolated polynucleotides each independently comprising, consisting of, or consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOs:2 to 65, and in certain of these embodiments the set is selected from a) a set of polynucleotides representing each of SEQ ID NOs:2-9, b) a set of polynucleotides representing each of SEQ ID NOs:10-17, c) a set of polynucleotides representing each of SEQ ID NOs:18-25, d) a set of polynucleotides representing each of SEQ ID NOs:26-33, e) a set of polynucleotides representing each of SEQ ID NOs:34-41, f) a set of polynucleotides representing each of SEQ ID NOs:42-49, g) a set of polynucleotides representing each of SEQ ID NOs:50-57, h) a set of polynucleotides representing each of SEQ ID NOs:58-65, i) a set of polynucleotides representing each of SEQ ID NOs:2-9 and 50-57, j) a set of polynucleotides representing each of SEQ ID NOs:10-17 and 58-65, k) a set of polynucleotides representing each of SEQ ID NOs:18-25 and 34-41, l) a set of polynucleotides representing each of SEQ ID NOs:26-33 and 42-49, m) a set of polynucleotides representing each of SEQ ID NOs:2-9, 18-25, 34-41, and 50-57, n) a set of polynucleotides representing each of SEQ ID NOs:10-17, 26-33, 42-49, 58-65; and o) a set of polynucleotides representing each of SEQ ID NOs:2-65.
Provided herein in certain embodiments are methods of preparing a target nucleic acid for sequencing comprising the steps of: a) ligating a 3′ adaptor to the target nucleic acid, wherein the 3′ adaptor consists of the nucleotide sequence of SEQ ID NO:66 or is an extended 3′ adaptor comprising the nucleotide sequence of SEQ ID NO:66 with one or more nucleotides added to the 5′ end; b) ligating a 5′ adaptor to the 3′ adaptor-target nucleic acid complex of step (a), wherein the 5′ adaptor consists of the nucleotide sequence of SEQ ID NO:1 or is an extended 5′ adaptor comprising the nucleotide sequence of SEQ ID NO:1 with one or more nucleotides added to the 3′ end; c) reverse transcribing the 3′ adaptor-target nucleic acid-5′ adaptor ligation product of step (b); d) PCR amplifying the reverse transcribed ligation product of step (c); and e) gel purifying the amplified target nucleic acid; provided that at least one of steps (a) and (b) must utilize an extended 3′ or 5′ adaptor, respectively. In certain of these embodiments, the target nucleic acid is a DNA or RNA molecule, and in certain of these embodiments the target nucleic acid is a smRNA. In certain embodiments, the methods further comprise a step of sequencing the gel purified target nucleic acid of step (e). In certain embodiments, the extended 5′ adaptor comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:2 to 65. In certain embodiments, more than one 3′ adaptor and/or more than one 5′ adaptor is used for steps (a) and (b), respectively. In certain of these embodiments, step (b) utilizes a pool of 5′ adaptors each independently comprising, consisting of, or consisting essentially of the nucleotide sequences of SEQ ID NOs:2 to 65.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Diagram of Illumina v1.5 smRNA preparation and sequencing protocol.
FIG. 2: Heatmap of clustered normalized reads of miRNA population for 64 customized 5′ adaptors. Total reads of each adaptor were normalized to a constant and each miRNA was scaled up according to the ratio of constant to the total reads. Normalized reads were Log2 transformed and the value was used to cluster miRNAs as complete linkage and generate the heatmap.
FIG. 3: ACGU distribution of the first three bases in all human miRNAs documented in miRBase 15.
FIG. 4: miRNA expression variation as measured using designed 8×8 adaptor pools (P1 to P8) and randomized adaptor pools (R1 to R8).
FIG. 5: Pearson correlation of designed 8×8 adaptor pools (P1 to P8) versus randomized adaptor pools (R1 to R8).
FIG. 6: Heatmap from Test 2. Lanes 2 and 6, duplicate run of 8'8 pools; Lane 3, 16×4 pools; Lane 4, 32×2 pools; Lane 5: 64×1 pool. Total reads of miRNAs in different lanes were normalized and log2 transformed to generate the heatmap.
FIG. 7: Normalized, log2 transformed miRNA read counts from Lane 2 (X-axis) and Lane 6 (Y-axis) of Test 2.
FIG. 8: Pearson correlation among adaptor pools in Test 2. Variation between adaptor pools was greatly reduced in the 16×4 pools (P17, P28, P35, and P46) versus the 8×8 pools (P1 to P8).
FIG. 9: A. Reproducibility among eight adaptor pools. Normalized, log2 transformed miRNA read counts from Lane 7 of Test 2 using breast RNA were plotted by adaptor pools. B. Heatmap of breast RNA eight adaptor pooling results.
FIG. 10: Reproducibility among 16, 32, and 64 adaptor pools. Normalized, log2 transformed miRNA read counts from Test 2 using breast RNA were plotted by adaptor pools.
FIG. 11: Northern blot analysis of miR-21, -1, -126, -205, and -101 in human RNA pool and human breast RNA. U6 snRNA was used as RNA loading control. Lane 0: RNA decade marker; Lane 1: human RNA pool; Lane 2: breast RNA.
FIG. 12: Heatmaps of 8×8 pools versus 16×4 pools versus 32×2 pools in Test 1. These results indicate that index pooling reduced miRNA expression variation.
FIG. 13: Re-analyzed data of Test 1. Data was reorganized by 16×4 adaptor pools. These results indicate that miRNA expression between designed pools shows less variation.
FIG. 14: Re-analyzed data of Test 1. Data was reorganized by 32×2 adaptor pools. These results indicate that miRNA expression between pools shows even less variation.



DETAILED DESCRIPTION
The following description of the invention is merely intended to illustrate various embodiments of the invention. As such, the specific modifications discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein.
Illumina's v1.5 smRNA preparation protocol (“Illumina protocol”) utilizes five main steps: 1) ligation of the 3′ adaptor to smRNA, 2) ligation of the 5′ adaptor to smRNA, 3) reverse transcription of smRNA with both adaptors to cDNA, 4) PCR amplification of the cDNA, and 5) smRNA gel purification. The default 3′ adaptor, which is modified to target smRNAs with a 3′ hydroxyl group, corresponds to the surface-bound amplification primer and is required for reverse transcription. The default 5′ adaptor, which has the nucleotide sequence set forth in SEQ ID NO:1, is required for amplification of smRNA fragments.
smRNA libraries prepared by the Illumina protocol are loaded onto single-read flowcells for cluster generation. During this process, the smRNA samples are bound to complementary adaptor oligonucleotides grafted onto the surface of an Illumina Genome Analyzer flow cell. These complementary adaptor oligonucleotides bind to either the 5′ or 3′ adaptors. Unlabeled nucleotides are added to initiate a solid-phase bridge amplification, which results in the formation of double-stranded bridges. The double-stranded molecules are denatured to generate single-stranded templates anchored to the substrate, followed by amplification to generate dense clusters of double-stranded DNA. Sequencing by synthesis (SBS) is then performed to ascertain the sequences of the smRNAs.
One drawback of the Illumina protocol is that the bridge amplification step requires both the 5′ and 3′ adaptors to be complementary to the complementary adaptor oligonucleotides on the flowcell surface. This creates a problem using bases within the sequences of either adaptor for barcoding.
Due to heterogeneity of the 3′ends of smRNAs, the fact that SBS is more likely to generate errors close to the end of the short reads (Wu 2007; Tian 2010), and the fact that the Illumina protocol specifically requires the pre-adenylated 5′ end of the 3′ adaptor (which is expensive to make), the 3′ end of the 5′ adaptor is the choice for multiplexing in the same flowcell for sequencing smRNAs of about 17 to 32 nts in length, such as piRNA, tRNA halves, and snoRNA derived smRNAs (Riedmann 2010; Thomson 2009; Pederson 2010; Ender 2008; Taft 2009).
As disclosed herein, the Illumina protocol was modified by adding three nucleotides (nt) at the 3′ end of the default 5′ adaptor for multiplexing. 64 different three nt sequences were utilized, representing all possible three nt combinations. The resultant customized 5′ adaptors were compared to the default Illumina 5′ adaptor for their efficacy in identifying miRNAs from various tissue samples using the Illumina protocol. Significant read biases were observed for each of the adaptor sequences. This kind of bias in smRNA digital gene expression profiling has been reported previously (Linsen 2009). To overcome these biases, the customized 5′ adaptors were combined into eight pools of eight adaptors each, four pools of 16 adaptors each, two pools of 32 adaptors each, and one pool of 64 adaptors. Pooling was based on the cluster data for the 64 customized 5′ adaptors. The last three nts of the customized 5′ adaptors were all found to influence the sequencing results (FIG. 2). Analysis of the pool data showed that pools containing 16, 32, or 64 customized 5′ adaptors minimized results bias, and were sufficient to identify all highly expressed miRNAs in various tissue samples (FIGS. 4, 5, and 12-14). Most importantly, subsequent comparison of miRNA sequencing of the same miRNA sample showed that the pooled customized 5′ adaptors dramatically reduced result bias as compared to the default 5′ adaptor. Use of all pools (8, 16, 32, and 64) resulted in a significant improvement in miRNA population coverage versus the use of the default adaptor alone, and resulted in identification of 893 of the 1,100 human mature miRNA/miRNA* sequences (about 81%) documented in miRBase 15. To further verify the superiority of the pooled adaptors to the default Illumina 5′ adaptor, the eight adaptor pools and the default Illumina 5′ adaptor were used to sequence miRNA from human breast tissue. The eight adaptor pools produced results that were more consistent with Northern blotting results than those obtained using the default adaptor (FIG. 11).
In summary, the results disclosed herein reveal a significant pitfall in the use of the default 5′ adaptor in the Illumina protocol. Sequence results obtained using this adaptor are biased by the short nucleotide fragments being sequenced, and therefore may not reflect the true biological composition of a sequence sample. Coverage and abundance are crucial to the evaluation of miRNA biological function and to the utilization of miRNAs as biomarkers in a clinical setting.
The compositions and methods disclosed herein can be used to overcome various shortcomings associated with Illumina and other next generation sequencing technologies that utilize adaptor sequences ligated to a sequence of interest, and to provide improved sequencing results for target nucleic acids. These target nucleic acids may be DNA or RNA molecules, including for example smRNA molecules. In certain embodiments, the target nucleic acids may be part of a target nucleic acid sample containing two or more target nucleic acids. In these embodiments, the target nucleic acid sample may comprise two or more copies of a single target nucleic acid, or it may comprise one or more copies of two or more different target nucleic acids. For example, a target nucleic acid sample may comprise one or more RNA molecules, DNA molecules, or a mixture thereof (such as a genomic nucleic acid sample).
Provided herein in certain embodiments are isolated polynucleotides for use in identifying, tagging, and sequencing a target nucleic acid. The target nucleic acid may be a DNA molecule or an RNA molecule, and in certain embodiments the target nucleic acid may be a smRNA molecule. In certain embodiments, the isolated polynucleotides consist of or consist essentially of the nucleotide sequence of the default Illumina 5′ adaptor (SEQ ID NO:1) with one or more nucleotides added to the 3′ end. In certain of these embodiments, the isolated polynucleotides consist of or consist essentially of the nucleotide sequence of SEQ ID NO:1 plus one, two, three, four, five, six, seven, eight, or more than eight nucleotides added to the 3′ end. In certain of these embodiments, the isolated polynucleotides comprise, consist of, or consist essentially of a nucleotide sequence as set forth in any of SEQ ID NOs:2-65.
Provided herein in certain embodiments are isolated polynucleotides for use in identifying, tagging, and sequencing a target nucleic acid. The target nucleic acid may be a DNA molecule or an RNA molecule, and in certain embodiments the target nucleic acid may be a smRNA molecule. In certain embodiments, the isolated polynucleotides consist of or consist essentially of the nucleotide sequence of the default Illumina 3′ adaptor (SEQ ID NO:66) with one or more nucleotides added to the 5′ end. In certain of these embodiments, the isolated polynucleotides consist of or consist essentially of the nucleotide sequence of SEQ ID NO:66 plus one, two, three, four, five, or more than five nucleotides added to the 5′ end.
Provided herein in certain embodiments are compositions comprising one or more of the isolated polynucleotides provided herein. In certain embodiments, these compositions comprise four or more, eight or more, 16 or more, 24 or more, 32 or more, 40 or more, 48 or more, 56 or more, or 64 or more of the isolated polynucleotides provided herein. In certain embodiments, the compositions comprise isolated polynucleotides comprising, consisting of, or consisting essentially of each of the nucleotide sequences of 1) SEQ ID NOs:2-9; 2) SEQ ID NOs:10-17; 3) SEQ ID NOs:18-25; 4) SEQ ID NOs:26-33; 5) SEQ ID NOs:34-41; 6) SEQ ID NOs:42-49; 7) SEQ ID NOs:50-57; 8) SEQ ID NOs:58-65; 9) SEQ ID NOs:2-9 and 50-57; 10) SEQ ID NOs:10-17 and 58-65; 11) SEQ ID NOs:18-25 and 34-41; 12) SEQ ID NOs:26-33 and 42-49; 13) SEQ ID NOs:2-9, 18-25, 34-41, and 50-57; 14) SEQ ID NOs:10-17, 26-33, 42-49, 58-65; and 15) SEQ ID NOs:2-65. In certain embodiments, these kits further comprise instructions for use.
Provided herein in certain embodiments is the use of one or more of the isolated polynucleotides or compositions provided herein for the identification, tagging, sequencing, and/or preparation for sequencing of one or more target nucleic acids. The target nucleic acids may be DNA molecules or RNA molecules, and in certain embodiments the target nucleic acids may be smRNA molecules.
Provided herein in certain embodiments are kits comprising one or more of the polynucleotides provided herein. In certain embodiments, these kits comprise four or more, eight or more, 16 or more, 24 or more, 32 or more, 40 or more, 48 or more, 56 or more, or all 64 of the isolated polynucleotides provided herein. In certain embodiments, the kits comprise isolated polynucleotides comprising, consisting of, or consisting essentially of each of the nucleotide sequences of 1) SEQ ID NOs:2-9; 2) SEQ ID NOs:10-17; 3) SEQ ID NOs:18-25; 4) SEQ ID NOs:26-33; 5) SEQ ID NOs:34-41; 6) SEQ ID NOs:42-49; 7) SEQ ID NOs:50-57; 8) SEQ ID NOs:58-65; 9) SEQ ID NOs:2-9 and 50-57; 10) SEQ ID NOs:10-17 and 58-65; 11) SEQ ID NOs:18-25 and 34-41; 12) SEQ ID NOs:26-33 and 42-49; 13) SEQ ID NOs:2-9, 18-25, 34-41, and 50-57; 14) SEQ ID NOs:10-17, 26-33, 42-49, 58-65; and 15) SEQ ID NOs:2-65. In certain embodiments, these kits further comprise instructions for use.
Provided herein in certain embodiments are methods for improving the Illumina protocol by using one or more of the isolated polynucleotides provided herein in place of the default Illumina 5′ adaptor of SEQ ID NO:1 and/or the default Illumina 3′ adaptor of SEQ ID NO:66. In certain embodiments, more than one of the isolated polynucleotides provided herein are utilized such that a pool of 5′ or 3′ adaptors is present in the RNA adaptor ligation mixture. In certain of these embodiments, the 5′ and/or 3′ adaptor pool comprises eight or more, 16 or more, 24 or more, 32 or more, 40 or more, 48 or more, 56 or more, or 64 or more of the isolated polynucleotides provided herein. In certain of these embodiments, the 5′ adaptor pool comprises a combination of isolated polynucleotides comprises, consisting of, or consisting essentially of each of the nucleotide sequences of 1) SEQ ID NOs:2-9; 2) SEQ ID NOs:10-17; 3) SEQ ID NOs:18-25; 4) SEQ ID NOs:26-33; 5) SEQ ID NOs:34-41; 6) SEQ ID NOs:42-49; 7) SEQ ID NOs:50-57; 8) SEQ ID NOs:58-65; 9) SEQ ID NOs:2-9 and 50-57; 10) SEQ ID NOs:10-17 and 58-65; 11) SEQ ID NOs:18-25 and 34-41; 12) SEQ ID NOs:26-33 and 42-49; 13) SEQ ID NOs:2-9, 18-25, 34-41, and 50-57; 14) SEQ ID NOs:10-17, 26-33, 42-49, 58-65; and 15) SEQ ID NOs:2-65.
Provided herein in certain embodiments are methods for preparing a target nucleic acid for sequencing comprising the steps of 1) ligating a 3′ adaptor to the target nucleic acid; 2) ligating a 5′ adaptor to the 3′ adaptor-target nucleic acid complex; 3) reverse transcribing the 3′ adaptor-target nucleic acid-5′ adaptor ligation product, 4) PCR amplifying the reverse transcribed ligation product to amplify target nucleic acids bound to adaptor molecules on both ends; and 5) gel purifying the amplified target nucleic acid, wherein one or both of the 3′ and 5′ adaptors are isolated polynucleotides as provided herein. In certain embodiments, the target nucleic acid is a smRNA molecule. In these embodiments, the methods comprise the steps of 1) ligating a 3′ adaptor to the smRNA; 2) ligating a 5′ adaptor to the 3′ adaptor-smRNA complex; 3) reverse transcribing the 3′ adaptor-smRNA-5′ adaptor ligation product, 4) PCR amplifying the reverse transcribed ligation product to amplify smRNAs bound to adaptor molecules on both ends; and 5) gel purifying the amplified smRNA, wherein one or both of the 3′ and 5′ adaptors are isolated polynucleotides as provided herein. As one example, in certain embodiments the 5′ adaptor comprises the nucleotide sequence of SEQ ID NO:1 with one or more nucleotides added to the 3′ end, and/or the 3′ adaptor comprises the nucleotide sequence of SEQ ID NO:66 with one or more nucleotides added to the 5′ end. In certain embodiments, a pool of more than one 5′ or 3′ adaptor is utilized for the ligation step. For example, the ligation step may utilize a pool of adaptors comprising eight or more, 16 or more, 24 or more, 32 or more, 40 or more, 48 or more, 56 or more, or 64 or more of the isolated polynucleotides provided herein. In certain embodiments, the methods of target nucleic acid preparation disclosed herein further comprise the step of actually sequencing the target nucleic acid, for example using Illumina NGS technology.
In certain embodiments of the target nucleic acid preparation methods provided herein wherein the target nucleic acid is smRNA, the primary method steps are carried out according to the Illumina v1.5 smRNA preparation protocol, with the only difference being the substitution or supplementation of the Illumina default 5′ and/or 3′ adaptors with one or more of the isolated polynucleotides provided herein.
In certain embodiments of the target nucleic acid preparation methods provided herein wherein the target nucleic acid is smRNA, the 3′ ligation adaptor ligation step is carried out as follows. The 3′ ligation adaptor(s) is diluted in nuclease free water and mixed with the smRNA sample, and this mixture is incubated at 70° C. for two minutes. The mixture is placed on ice, then mixed with T4 RNL2 truncated reaction buffer (NEB), MgCl2, truncated T4 RNA ligase 2 (NEB), and RNase inhibitor and incubated at 22° C. for one hour. In certain embodiments, one or more oligonucleotides complementary to all or part of the 3′ ligation adaptor(s) are included in the ligation reaction mixture. In these embodiments, the oligonucleotides hybridize to the 3′ adaptors to prevent hybridization of the adaptors to smRNA. In certain of these embodiments, the oligonucleotide may be tagged to facilitate oligonucleotide removal following completion of the 3′ ligation adaptor ligation step. Suitable tags include, for example, magnetic beads or biotin. In certain embodiments, the oligonucleotides may comprise all or a portion of the sequence of the default Illumina PCR primers set forth in SEQ ID NOs:67 and 68.
In certain embodiments of the target nucleic acid preparation methods provided herein wherein the target nucleic acid is smRNA, the 5′ ligation adaptor ligation step is carried out as follows. The 5′ ligation adaptor(s) is pre-heated to 70° C. for two minutes, transferred to ice, and then added to the 3′ ligation mixture of the previous paragraph along with ATP and T4 RNA ligase. This mixture is incubated at 20° C. for one hour. In certain embodiments, one or more oligonucleotides complementary to all or part of the 5′ ligation adaptor(s) are included in the ligation reaction mixture. In these embodiments, the oligonucleotides hybridize to the 5′ adaptors to prevent hybridization of the adaptors to smRNA. In certain of these embodiments, the oligonucleotide may be tagged to facilitate oligonucleotide removal following completion of the 5′ ligation adaptor ligation step. Suitable tags include, for example, magnetic beads or biotin. In certain embodiments, the oligonucleotides may comprise all or a portion of the sequence of the default Illumina PCR primers set forth in SEQ ID NOs:67 and 68.
In certain embodiments of the target nucleic acid preparation methods provided herein wherein the nucleic acid is smRNA, the reverse transcription step is carried out as follows. The ligated smRNA sample is mixed with SRA RT primer (Illumina), centrifuged briefly, heated at 70° C. for two minutes, then placed on ice. First strand buffer (Invitrogen), dNTP mix, DTT, and RNase inhibitor are mixed together, then added to the primer-annealed ligated smRNA sample and heated at 48° C. for three minutes. SuperScript II Reverse Transcriptase (Invitrogen) is added to the mixture, followed by incubation at 44° C. for one hour.
In certain embodiments of the target nucleic acid preparation methods provided herein wherein the target nucleic acid is smRNA, the post-reverse transcription PCR amplification step is carried out as follows. Water, Phusion HF buffer (Illumina), primer GX1 (Illumina), primer GX2 (Illumina), dNTP mix, and Phusion DNA polymerase (Illumina) are mixed together, and the reverse transcribed smRNA sample is added to this mixture. PCR amplification is then performed using one 30 second cycle at 98° C., twelve cycles of 10 second at 98° C., 30 seconds at 60° C., and 15 seconds at 72° C., and a ten minute cycle at 72° C., followed by holding the sample at 4° C. In certain embodiments, the number of cycles may be adjusted up or down based on the amount of smRNA in the initial sample.
In certain embodiments of the target nucleic acid preparation methods provided herein wherein the target nucleic acid is smRNA, the target smRNA may undergo one or more additional steps before, during, or after the primary method steps. For example, the target smRNA may undergo one or more isolation, purification, and/or concentrating steps prior to the 3′ adaptor ligation step. Similarly, the target smRNA may undergo one or more amplification steps prior to the 3′ adaptor ligation step. In certain embodiments, the integrity of the target smRNA molecule may be evaluated before, during, or after any of the primary method steps.
A target nucleic acid for use in the methods disclosed herein may be obtained from any biological sample containing nucleic acids such as DNA, RNA, smRNA, or combinations thereof. The biological sample may be obtained from any species, including a mammalian species such as a human. In certain embodiments, the target nucleic acid sample is obtained from a bodily fluid such as blood or saliva. In other embodiments, the target nucleic acid sample is obtained from a solid tissue sample, including for example a biopsy sample. In certain embodiments wherein the target nucleic acid is an smRNA molecule, the smRNA molecule may be in a sample comprising more than 1 μg of total RNA, and in certain of these embodiments the smRNA sample comprises more than 2 μg, more than 4 μg, more than 6 μg, more than 8 μg, or more than 10 μg of total RNA. In certain embodiments, the smRNA sample comprises 1-10 μg of total RNA.
Provided herein in certain embodiments are methods of diagnosing a disease using the target nucleic acid preparation and sequencing methods provided herein. In these embodiments, nucleic acid samples from a subject are prepared and sequenced using the methods disclosed herein, then compared to one or more reference sequences representing nucleic acid sequences associated with a particular disease. For example, where the nucleic acid samples contain smRNA, the reference sequences may include bacterial or viral smRNA sequences. In these embodiments, identification of a nucleic acid sequence matching one of the reference sequences indicates the presence of a particular disease, including for example a bacterial or viral infection.
The term “comprising” as used herein, particularly with regard to a component of a composition or a step of a method, encompasses compositions and methods consisting of or consisting essentially of the component or step.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
EXAMPLES
Example 1
Evaluation of Three Nucleotide 5′ Adaptor Extensions
It has been reported previously that it is possible to add several nts to the 5′ adaptor tail for barcoding for miRNA profiling (Witten 2010; Cronn 2008). To evaluate the effect of three nt extensions, 64 different extension sequences (Table 1) were added to the 3′ end of the default Illumina 5′ adaptor (SEQ ID NO:1) to produce a series of 64 customized 5′ adaptors.







TABLE 1





Three nucleotide extensions


(SEQ ID NO: of resulting customized 5′ adaptor)






















AAA
AAU
AAC
AAG
GAA
GAU
GAC
GAG


(2)
(10)
(18)
(26)
(34)
(42)
(50)
(58)


UAU
UAC
UAG
UAA
AGU
AGC
AGG
AGA


(3)
(11)
(19)
(27)
(35)
(43)
(51)
(59)


AUC
AUG
AUA
AUU
ACC
ACG
ACA
ACU


(4)
(12)
(20)
(28)
(36)
(44)
(52)
(60)


UUG
UUA
UUU
UUC
UCG
UCA
UCU
UCC


(5)
(13)
(21)
(29)
(37)
(45)
(53)
(61)


GCA
GCU
GCC
GCG
GUA
GUU
GUC
GUG


(6)
(14)
(22)
(30)
(38)
(46)
(54)
(62)


CCU
CCC
CCG
CCA
CUU
CUC
CUG
CUA


(7)
(15)
(23)
(31)
(39)
(47)
(55)
(63)


CGC
CGG
CGA
CGU
CAC
CAG
CAA
CAU


(8)
(16)
(24)
(32)
(40)
(48)
(56)
(64)


GGG
GGA
GGU
GGC
UGG
UGA
UGU
UGC


(9)
(17)
(25)
(33)
(41)
(49)
(57)
(65)









For Test 1, the 64 customized 5′ adaptors were individually ligated with smRNA-3′adaptor molecules and smRNA libraries from eight adaptors pooled together for cluster generation and SBS. smRNA preparation was carried out using the Illumina manufacturer's protocols (Preparing Samples for Small RNA Sequencing Using the Alternative v1.5 Protocol, Small RNA sample prep Kit# FC-102-1009, Illumina Inc.) with minor optimization. Equal amounts of 20 human tissue RNAs from Ambion human total RNA survey panel (AM6000) were mixed as human total RNA pool in order to balance smRNA population and mimic the abundance and 3′ and 5′ heterogeneity of smRNA sequences. RNA in the pool was derived from adipose, bladder, brain, cervix, colon, esophagus, heart, kidney, liver, lung, ovary, placenta, prostate, skeletal muscle, small intestine, spleen, testes, thymus, thyroid, and trachea tissue. The RNA pool was used for smRNA library construction, cluster generation, and SBS. 1.0 μg of pooled total RNA was ligated to the default Illumina 3′ adaptor (SEQ ID NO:66) with T4 RNA ligase 2 and truncated (New England BioLabs) at 22 ° C. for 1 hour; The 3′ adaptor-miRNA ligation was then ligated to the default Illumina 5′ adapter, one of the 64 customized 5′ adaptors, or an 8, 16, 32, or 64 adaptor pool of customized 5′ adaptors (0.5 μl of 5 μM per reaction) with T4 RNA ligase I (New England BioLabs) at 20° C. for 1 hour. The resultant smRNA library was reverse transcribed using GX1 (SEQ ID NO:67) as RT primer, then subjected to PCR amplification for 12 cycles using the primers GX1 and GX2 (SEQ ID NO:68). Amplified smRNAs were gel purified using a 6% TBE PAGE gel with size selection (for targeted smRNA of 17-32 nt). The purified library was quantified using qPCR with the forward primer of SEQ ID NO:69 and the reverse primer of SEQ ID NO:70, and the quantified denatured miRNA library was loaded in 1 ml hybridization buffer to a final DNA concentration of 8 pM and used for single read flow cell cluster generation. 42 cycle (42 nt) sequencing was performed using Illumina Genome Analyzer II (GAII).
Raw reads from Test 1 shown in Table 2 (set forth in the file “54435.8095.US00.txt” submitted herewith on CD-R), and the test results are summarized in Table 3. Total reads for each adaptor were normalized to a constant, and each miRNA was scaled up according to the ratio of constant to the total reads. Log2 transformed normalized reads were clustered and used to generate a heatmap (FIG. 2). Serious result biases were introduced by both the customized 5′ adaptors and the default 5′ adaptor. There were biases in the number of total reads, percentage of reads that were aligned to corresponding genomes, distribution of smRNAs to different categories, coverage of different species in the same smRNA population, and abundance of individual smRNAs. Read counts for some individual miRNAs were different by as much as 200-fold.
To eliminate the possibility that the smRNA reads processing software might be producing the bias, reads were profiled using different miRNA profiling software systems implemented with different data analysis algorithms. Reads from the 8-pool adaptors from lane 1 of Test 1 were profiled using miRExpress and Novoalign.
For Novoalign data processing, image analysis and base calling analysis were performed using the Illumina Genome Analysis Package (OLB v1.6 and CASAVA v1.6). The base calling analysis did not apply the Illumina default chastity filter, which discards about 30 to 40% of the reads with low quality from the first 25 bases being sequenced and generated 42-nt long reads in FASTQ format. Quality control of the reads was done during alignment by Novoalign software that filters out low quality reads by base quality score. Comparison studies showed that this Novoalign filters performed similarly to the Illumina default chastity filter. Reads were separated using Novobarcode software (http://www.novocraft.com), and aligned to human genome hg18 (NCBI build 36.1) using Novoalign software (http://www. novocraft.com) with default settings but for the following modifications: an aligned read requires a minimum of 16 nts in length with good base quality after 3′ adapter trimming and a perfect match to the reference genome. A mapping table was created using the human miRNA mature sequences from miRBase 15 and aligned back to the hg18 genome afterwards. If a read was able to be aligned to multiple loci, it was randomly assigned to one locus. To summarize the expression levels of smRNAs, reads of a smRNA falling into the mapped smRNA regions within five base extensions were treated as valid reads and counted. If a smRNA could be mapped to multiple genome loci, the counts at each locus were summed as the total number of reads for that smRNA.
For statistical analysis of the miRNA expression levels, total count of miRNAs in each barcoded sample was scaled to a constant (1.7 million) and log2 transformed with an offset of one. MiRNAs with normalized reads over 6 in at least 10% of the samples were kept for subsequent expression correlation efficiency analysis. Hierarchical clustering with one-Pearson correlation as distance measurement and complete linkage was conducted with Cluster 3.0 and viewed in Java TreeView (de Hoon 2004; Saldanha 2004).
miRExpress software analyzed miRNAs by matching reads directly to pre-miRNA or mature miRNAs sequences (Wang 2009). The default setting was used to count reads that could perfectly match to pre-miRNAs.
Although the rank of miRNAs by read counts in miRExpress and Novoalign was not identical, read counts and abundance rank were comparable between the two programs (Table 4). These results establish that the data analysis algorithm is not a major cause for the sequencing bias.
Table 5 compares the results for the top 20 miRNAs with both the default 5′ adaptor and the customized 5′ adaptors. These results suggest that the default 5′ adaptor is incapable of sequencing certain miRNAs (e.g., miR-1, -145, -215, -126 and -451), but favors others (e.g., miR-20a, -17, -155, -1246 and -146a).








TABLE 5







Sorted by



default 5′ adaptor
Sorted by average











Pool-

of adaptors pool












miRNA
avg
Default
miRNA
Pool-avg
Default















hsa-miR-21
339251
1852326
hsa-miR-143
1984995
17640


hsa-miR-378
178769
1201524
hsa-miR-1
429851
39


hsa-let-7f
211659
510653
hsa-miR-21
339251
1852326


hsa-miR-30e
50344
238220
hsa-let-7a
221170
182815


hsa-miR-30d
68551
235117
hsa-let-7f
211659
510653


hsa-miR-101
57845
194516
hsa-miR-378
178769
1201524


hsa-let-7a
221170
182815
hsa-miR-126
169586
2


hsa-miR-
69620
169524
hsa-miR-30a
140659
425


142-3p


hsa-miR-
77003
167654
hsa-miR-26a
137699
23823


148a


hsa-miR-20a
9939
138720
hsa-miR-24
123388
1815


hsa-miR-155
3910
118345
hsa-miR-
77003
167654





148a


hsa-miR-103
23391
97269
hsa-miR-192
71314
12660


hsa-miR-17
8244
75571
hsa-miR-
69620
169524





142-3p


hsa-let-7g
47101
72910
hsa-miR-30d
68551
235117


hsa-miR-
38710
67006
hsa-miR-
67342
51686


140-3p


146b-5p


hsa-miR-
67342
51686
hsa-miR-145
67237
40


146b-5p


hsa-miR-191
22303
49626
hsa-miR-29a
65488
40124


hsa-miR-
6148
49235
hsa-miR-101
57845
194516


1246


hsa-miR-
8939
45680
hsa-miR-215
57179
142


146a


hsa-miR-29a
65488
40124
hsa-miR-451
56674
5









Neither the distribution of the first three nts in the human miRNAs (FIG. 3) nor the secondary structures and thermal dynamic properties of the 64 adaptors (Table 3, columns 2, 3, and 9) were found to have a reasonable correlation with the result bias. Interestingly, the clustered Test 1 results revealed that all three added nts influenced the results. This clustering result was roughly divided into eight groups (FIG. 2). Based on these eight groups, the 64 customized adaptors were organized into eight designed pools of eight adaptors each (P1-P8) with balanced bases at each position. These 8×8 pools were merged to form four pools of 16 adaptors each (P17, P28, P35, and P46), the 16×4 pools were merged to form two pools of 32 adaptors each (P1357 and P2468), and the two 32×2 pools were merged to form a single pool of 64 adaptors (Pall). The pooling strategy is summarized in Table 6.















TABLE 6








P1
P2
P3
P4
P5
P6
P7
P8





 8 × 8
AAA
AAU
AAC
AAG
GAA
GAU
GAC
GAG


pools
UAU
UAC
UAG
UAA
AGU
AGC
AGG
AGA



AUC
AUG
AUA
AUU
ACC
ACG
ACA
ACU



UUG
UUA
UUU
UUC
UCG
UCA
UCU
UCC



GCA
GCU
GCC
GCG
GUA
GUU
GUC
GUG



CCU
CCC
CCG
CCA
CUU
CUC
CUG
CUA



CGC
CGG
CGA
CGU
CAC
CAG
CAA
CAU



GGG
GGA
GGU
GGC
UGG
UGA
UGU
UGC















P17
P28
P35
P46





16 × 4
P1
P2
P3
P4


pools
P7
P8
P5
P6













P1357
P2468





32 × 2
P17
P28


pools
P35
P46












Pall





64 × 1
P1357


pool
P2468









Correlation efficiency showed that the designed pools of 8 adaptors provided significantly more compact results than randomized pools of 8 adaptors, with the designed pools showing less miRNA expression variation than the randomized pools (FIGS. 4 and 5). Pooled results from Test 1 indicate that index pooling reduced miRNA expression variation, with larger index pools resulting in less miRNA expression bias among same size pools (FIG. 12-14).
For Test 2, the designed adaptor pools were used for 5′ adaptor ligation to smRNA-3′adaptors. This approach allowed for the use of one group of 8, 16, 32, or 64 adaptors to barcode one sample. Consistent results were observed across all the adaptor pools (FIGS. 6 and 7, Table 7) The use of pools containing 16 adaptors was sufficient to minimize results bias (FIGS. 8-10), and miRNA expression measurement between the 16, 32, and 64 adaptor pools showed about 99% correlation (FIG. 6). Correlation efficiency using the 16 adaptor pool to run four samples per flowcell versus running one sample was about 0.99. Only about 10% of miRNAs were undetectable in the four sample run compared to the one sample run (Table 8). The 10% missed were typically less abundant miRNAs. Therefore, the use of 16 adaptor pools to barcode four samples per flowcell is sufficient to identify all highly expressed miRNAs with an expression measurement that is comparable to that obtained with one sample per lane.













TABLE 8





16 × 4


P17 in
P28 in
P35 in
P46 in


pool
miRBase
All in lane
Lane 4-
Lane 4-
Lane 4-
Lane 4-


results
15
4-Test 2
Test 2
Test 2
Test 2
Test 2







Unique
1100
763
666
664
678
663


miRNAs









Comparison of Test 2 data showed the profiling coverage of miRNAs increased by almost 100% using the pooled adaptors versus the default 5′ adaptor (Table 9).













TABLE 9








Default
64 × 1
32 × 2
16 × 4
8 × 8 pool
8 × 8 pool



adaptor
pool
pool
pool
(Lane 2)
(Lane 6)








Raw read
miRNA coverage measured by unique miRNA sequences


counts
(1100 in miRBase 15)
















≧1
461
776
760
763
774
769


≧5
348
636
643
627
625
645


≧10
297
573
583
573
575
579


≧100
189
379
383
364
373
382


≧1000
 93
184
184
171
174
179









Reads from all runs in both Test 1 and Test 2 showed that 893 miRNAs were detected (Table 10). This represents about 81% of the 1,100 human mature miRNA/miRNA* sequences documented in miRBase 15.









TABLE 10






miRNA coverage measured




by unique miRNA sequences


miRNA raw read counts
(Test 1 + Test 2)
By %

















≧1
893
81.18


≧5
821
74.64


≧10
776
70.55


≧100
603
54.82


≧1000
407
37.00









The low coverage and bias of the Illumina default adaptor may play a role in detecting low abundance miRNA and miRNA*. The miRNA* family are classified as low abundance strands from the same precursor miRNA. However, in the experiments disclosed herein, the abundance was reversed for 21 pairs of miRNA/miRNA*, with 28 miRNAs detected at less than 10 times the abundance of their corresponding miRNA* (Table 11).
To validate the pooling strategy, breast tissue total RNA (#AM6952, Table 7) and human pooled total RNA was sequenced using the eight adaptor pools or the default Illumina 5′ adaptor. The results showed consistent performance among the eight adaptor pools (FIG. 9). The relative abundance of miR-21, -1, -126, -101, and -205 in breast RNA and pooled human tissue RNA as measured by normalized read counts is set forth in Table 12. The eight adaptor pools were effective at identifying all five miRNAs in both tissue samples, while the default adaptor over-reported two (miR-21 and miR-101) and was largely ineffective at identifying the other three (miR-1, miR-126, and mi-R205).










TABLE 12





RNA source
Human RNA pool
Human RNA pool
Breast RNA


5′ Adaptor
Default
8 × 8 pool
8 × 8 pool


















hsa-miR-21
1911436
368479
488594


hsa-miR-1
40
450370
2909


hsa-miR-126
2
175101
222838


hsa-miR-101
200723
61050
77431


hsa-miR-205
0
5408
90397









Next, expression levels of miR-21, -1, -126, -101, and -205 in breast RNA versus pooled human tissue RNA were evaluated by Northern blot as previously described (Sun 2009). 20 μg of human pooled total RNA or breast total RNA were loaded onto a 12% PAGE/8M urea gel. Northern blot results were consistent with the eight adaptor pool sequencing results from both breast RNA and pooled human RNA (FIG. 11). However, the Northern blot results were not consistent with the default adaptor sequencing results from pooled RNA tissue. These results further verify the superiority of miRNA tagging and sequencing using the pooled adaptors versus the default Illumina adaptor.
As stated above, the foregoing are merely intended to illustrate the various embodiments of the present invention. As such, the specific modifications discussed above are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein. All references cited herein are incorporated by reference as if fully set forth herein.
REFERENCES
1. Bartel, Cell 136:215 (2009)
2. Chiang et al., Genes Dev 24:992 (2010)
3. Cronn et al., Nucl Acids Res 36:e122 (2008)
4. Cummins et al., Proc Natl Acad Sci USA 103:3687 (2006)
5. de Hoon et al., Bioinformatics 20:1453 (2004)
6. Ender et al., Mol Cell 32:519 (2008)
7. Galasso et al., Genome Med 2:12 (2010)
8. Goff et al., PLoS One 4:e7192 (2009)
9. Griffiths-Jones, Curr Protoc Bioinformatics Chapter 12, Unit 12.9.1 (2010)
10. Hafner et al., Methods 44:3 (2008)
11. Kim et al., Nat Rev Mol Cell Biol 10:126 (2009)
12. Lagos-Quintana et al., Science 294:853 (2001)
13. Lau et al., Science 294:858 (2001)
14. Lee et al., Cell 75:843 (1993)
15. Lee et al., Science 294:862 (2001)
16. Levin et al., Nat Methods 7:709 (2010)
17. Linsen et al., Nat Methods 6:474 (2009)
18. Lu et al., Science 309:1567 (2005)
19. Lu et al., Methods 43:110 (2007)
20. Nagpal et al., Curr Mol Med 10:503 (2010)
21. Pasquinelli et al., Nature 408:86 (2000)
22. Pederson, RNA 16:1865 (2010)
23. Pfeffer et al., Curr Protoc Mol Biol Chapter 26, Unit 26.4 (2005)
24. Reinhart et al., Nature 403:901 (2000)
25. Riedmann & Schwentner, RNA Biol 7:133 (2010)
26. Saldanha, Bioinformatics 20:3246 (2004)
27. Sun et al., RNA 15:1640 (2009)
28. Taft et al., RNA 15:1233 (2009)
29. Tang et al., Nat Protoc 5:516 (2010)
30. Thomson & Lin, Annu Rev Cell Dev Biol 25:355 (2009)
31. Tian et al., BMC Bioteclmol 10:64 (2010)
32. Wang et al., BMC Bioinformatics 10:328 (2009)
33. Wightman et al., Cell 75:855 (1993)
34. Witten et al., BMC Biol 8:58 (2010)
35. Wu et al., PLoS One 2:e1020 (2007)















TABLE 3












Correlation
Count of



# of





of miRNA
miRNAs



secondary





with
with same



structures





average # of
1st three



predicted



# of reads that

miRNA in
nts as the


Last 3 nts
by
# of G and

# of reads that
can be aligned
% of reads
all adaptors
last 3 nts of


of custom
QuikFold
C nts in
# of total
passed filter of
to human
that passed
(linear
the custom


adaptor
3.0
adaptor
reads
Novoalignment
genome 18
filter
scale)
adaptor























AAA
5
0
2,873,823
2,602,433
1,854,870
71.3%
0.9836
47


AAU
5
0
2,363,426
2,174,865
1,577,744
72.5%
0.9852
19


AAC
5
1
2,443,514
2,221,513
1,584,608
71.3%
0.9846
20


AAG
5
1
2,294,143
1,650,306
1,072,384
65.0%
0.9848
21


ACA
5
1
2,659,638
2,466,420
2,065,694
83.8%
0.9716
11


ACU
5
1
1,865,696
1,748,929
1,422,241
81.3%
0.9691
31


ACC
5
2
2,427,509
2,022,047
1,489,387
73.7%
0.9844
12


ACG
5
2
2,141,983
1,596,426
1,264,670
79.2%
0.9899
5


AUA
5
0
2,943,442
2,767,906
2,086,642
75.4%
0.9886
12


AUU
5
0
1,819,682
1,715,885
1,324,440
77.2%
0.9810
8


AUC
5
1
2,280,279
2,150,454
1,464,065
68.1%
0.9663
21


AUG
5
1
2,005,752
1,663,633
1,226,855
73.7%
0.9853
14


AGA
7
1
1,866,123
1,496,581
1,092,992
73.0%
0.9891
23


AGC
5
2
4,100,291
2,246,985
1,615,537
71.9%
0.9845
15


AGU
6
1
2,266,066
2,096,199
1,493,172
71.2%
0.9894
13


AGG
3
2
2,186,275
482,034
319,862
66.4%
0.9905
38


UAA
1
0
2,186,275
482,034
319,862
77.8%
0.9855
32


UAU
1
0
3,689,221
3,421,227
2,703,636
79.0%
0.9853
19


UAC
1
1
3,075,174
2,744,694
2,193,399
79.9%
0.8707
17


UAG
1
1
3,090,672
2,617,560
1,817,026
69.4%
0.9793
28


UCA
1
1
2,830,564
2,587,389
1,998,448
77.2%
0.5842
24


UCU
1
1
1,900,004
1,577,456
1,148,322
72.8%
0.3917
24


UCC
1
2
1,733,729
1,531,841
1,131,036
73.8%
0.5377
24


UCG
1
2
3,255,088
2,672,867
1,860,778
69.6%
0.3742
12


UUA
3
0
2,064,236
1,908,719
1,541,108
80.7%
0.6899
13


UUU
3
0
2,869,685
2,596,616
2,071,204
79.8%
0.7896
19


UUC
3
1
2,415,109
2,267,877
1,768,381
78.0%
0.9945
25


UUG
3
1
2,367,315
2,098,963
1,685,140
80.3%
0.6990
17


UGA
1
1
615,726
506,892
366,514
72.3%
0.9819
38


UGC
1
2
2,500,231
2,034,397
1,530,145
75.2%
0.9846
21


UGU
2
1
2,778,109
2,528,513
1,826,787
72.2%
0.6044
31


UGG
2
2
2,295,023
1,279,694
897,849
70.2%
0.9784
43


CAA
7
1
2,085,449
1,950,978
1,443,135
74.0%
0.9846
36


CAU
7
1
2,577,649
2,446,969
1,844,074
75.4%
0.9865
14


CAC
7
2
2,154,266
1,979,033
1,505,862
76.1%
0.9886
15


CAG
7
2
2,980,868
2,160,044
1,528,713
70.8%
0.9818
24


CCA
7
2
1,947,457
1,750,754
1,376,395
78.6%
0.9865
16


CCU
7
2
1,877,313
1,701,980
1,260,847
74.1%
0.9864
19


CCC
7
3
1,765,335
1,417,864
989,471
69.8%
0.9830
13


CCG
9
3
1,579,577
1,307,675
1,032,027
78.9%
0.9909
4


CUA
7
1
1,846,184
1,721,094
1,294,698
75.2%
0.9280
14


CUU
7
1
2,883,730
2,695,491
2,070,122
76.8%
0.9830
14


CUC
8
2
2,912,793
2,526,398
1,849,228
73.2%
0.9913
27


CUG
9
2
3,648,901
3,127,550
2,478,456
79.2%
0.8337
34


CGA
3
2
3,174,936
2,740,749
1,975,732
72.1%
0.9906
3


CGC
8
3
3,233,287
2,237,951
1,629,380
72.8%
0.9894
5


CGU
8
2
3,043,880
2,731,024
2,038,981
74.7%
0.9942
5


CGG
1
3
3,543,206
659,906
461,155
69.9%
0.9502
12


GAA
4
1
4,601,179
4,100,429
2,849,364
69.5%
0.9879
15


GAU
3
1
4,017,562
3,718,452
2,681,945
72.1%
0.9914
5


GAC
4
2
3,865,704
3,282,978
2,272,584
69.2%
0.9910
5


GAG
3
2
4,079,254
2,397,622
1,564,181
65.2%
0.9872
9


GCA
7
2
3,348,917
2,892,130
2,310,215
79.9%
0.9460
13


GCU
7
2
3,741,695
3,506,723
2,667,236
76.1%
0.9348
11


GCC
7
3
3,236,164
2,627,147
1,752,991
66.7%
0.9554
7


GCG
7
3
4,033,566
2,305,468
1,704,092
73.9%
0.9737
4


GUA
2
1
3,131,513
1,847,377
824,577
44.6%
0.9191
2


GUU
8
1
3,347,839
3,036,625
2,037,159
67.1%
0.8761
6


GUC
1
2
3,048,292
2,591,672
1,619,189
62.5%
0.9666
6


GUG
4
2
2,472,107
1,896,957
1,195,374
63.0%
0.7264
23


GGA
2
2
3,092,035
2,550,386
1,405,444
55.1%
0.8136
6


GGC
3
3
3,190,015
1,377,083
911,604
66.2%
0.8605
10


GGU
2
2
2,475,086
1,979,666
1,212,320
61.2%
0.9597
7


GGG
3
3
4,653,050
1,637,713
977,341
59.7%
0.7433
19


























TABLE 4





Rank
miRNA
AAA
AAT
CTC
CTT
GGG
GTG
TCA
TCC
sum















miRExpress software

















1
hsa-miR-143
629047
596534
264042
259410
49059
100716
59682
21985
1980475


2
hsa-miR-378
36063
53471
42317
54587
16247
28919
126718
57710
416032


3
hsa-miR-1
57848
44332
39707
10420
71826
135691
29183
16259
405266


4
hsa-miR-21
56293
54594
30282
46590
12227
15423
73223
35705
324337


5
hsa-miR-30a
18225
21466
16951
22039
9302
13824
71561
25377
198745


6
hsa-let-7a
73073
55464
6234
8420
8281
10661
15513
5619
183265


7
hsa-let-7f
80747
61375
3623
6323
4201
9192
11917
4434
181812


8
hsa-miR-126
21192
17954
10190
13560
10448
25185
32341
8650
139520


9
hsa-miR-26a
18178
12543
15896
37425
6207
9273
16873
9698
126093


10
hsa-miR-148a
13804
11033
13926
20902
12155
5385
12584
2984
92773


11
hsa-miR-215
12043
10792
13911
6560
3232
17876
18218
5473
88105


12
hsa-miR-192
11751
11388
8808
6573
5710
9914
19675
6537
80356


13
hsa-miR-101
7582
8456
7582
10921
3174
6450
20984
10424
75573


14
hsa-miR-30d
9416
9847
7963
8538
4882
8059
15766
6778
71249


15
hsa-miR-10b
9009
10431
6283
7604
4005
4395
19467
9012
70206


16
hsa-miR-451
15361
10057
7584
7501
2597
2635
10609
6821
63165


17
hsa-miR-145
2744
2847
10661
9089
4226
7679
11457
8859
57562


18
hsa-miR-27b
8511
6649
7872
9957
3741
2958
9543
4668
53899


19
hsa-miR-140-3p
7200
6803
7626
10477
3511
3559
6379
5750
51305


20
hsa-miR-24
5128
3009
9244
3013
7118
9415
7554
4633
49114







Novoalign software

















1
hsa-miR-143
619600
588860
261006
257701
57043
99605
60485
21845
1966145


2
hsa-miR-1
58842
45204
39539
10656
74017
137450
29480
16454
411642


3
hsa-miR-21
56707
54428
30261
46984
12404
15381
73132
35673
324970


4
hsa-miR-378
34181
51292
40421
52081
16619
27345
119105
54877
395921


5
hsa-let-7f
81639
62336
3727
6472
4770
9475
12242
4524
185185


6
hsa-let-7a
72376
55159
6190
8391
8775
13750
15523
5614
185778


7
hsa-miR-30a
18195
21486
16923
22285
9600
13915
71336
25348
199088


8
hsa-miR-26a
17701
12617
15479
38064
6365
8978
16648
9446
125298


9
hsa-miR-126
20722
17592
9962
13332
10604
24737
31622
8487
137058


10
hsa-miR-24
5094
2979
9192
3026
7340
9417
7490
4594
49132


11
hsa-miR-148a
13547
10840
13693
20651
12760
5338
12467
2945
92241


12
hsa-miR-215
11939
13790
13676
6407
3774
17552
17856
5352
90346


13
hsa-miR-192
11557
11232
8651
6496
5648
9717
19311
6395
79007


14
hsa-miR-30d
9362
9801
7964
8616
4963
8177
15716
6767
71366


15
hsa-miR-101
7589
8328
7469
10798
3144
6385
20665
10339
74717


16
hsa-miR-146b-5p
6947
6492
3325
3180
8576
7638
10232
1814
48204


17
hsa-miR-145
2685
2797
10421
8912
4111
7447
11085
8628
56086


18
hsa-miR-451
15263
9893
7453
7354
2578
2577
10438
6706
62262


19
hsa-miR-10b
8838
10204
6145
7515
4054
4383
19157
8867
69163


20
hsa-miR-27b
8140
6394
7546
10720
3945
2848
9217
4493
53303


















TABLE 7








Human RNA pool




adaptors
Breast














RNA
Default
8 × 8
16 × 4
32 × 2
64 × 1
8 × 8
8 × 8


miRNA
(lane 1)
(lane 2)
(lane 3)
(lane 4)
(lane 5)
(lane 6)
(lane 7)

















hsa-let-7a
182815
207044
201770
228567
250250
229745
285909


hsa-let-7a*
191
183
156
229
243
178
321


hsa-let-7a-2*
0
10
14
22
19
13
8


hsa-let-7b
3732
38250
37832
43779
45636
42083
104331


hsa-let-7b*
7
168
150
202
204
167
305


hsa-let-7c
4493
38771
37083
42216
46566
42940
42483


hsa-let-7c*
1
2
4
2
5
3
6


hsa-let-7d
9918
4502
4469
5067
4961
4926
3779


hsa-let-7d*
698
958
929
1221
1170
1079
1078


hsa-let-7e
249
8159
7300
7306
6986
9357
6280


hsa-let-7e*
0
51
58
78
81
77
47


hsa-let-7f
510653
201473
192459
203236
227113
225547
159271


hsa-let-7f-1*
62
20
20
32
18
16
28


hsa-let-7f-2*
80
2
6
4
5
5
9


hsa-let-7g
72910
42837
43556
50078
53190
47164
53943


hsa-let-7g*
43
219
245
315
328
256
167


hsa-let-7i
5027
8012
7610
8665
8993
8867
9347


hsa-let-7i*
113
304
314
419
420
318
426


hsa-miR-1
39
404787
408176
485549
509267
450370
1790


hsa-miR-100
1
24727
23218
27907
27055
26815
6954


hsa-miR-100*
0
38
27
39
33
30
5


hsa-miR-101
194516
55392
50631
59094
59555
61050
47642


hsa-miR-101*
133
369
376
446
444
413
235


hsa-miR-103
97269
20956
17889
14087
12536
23100
8047


hsa-miR-103-2*
37
20
14
31
21
20
22


hsa-miR-103-as
0
0
0
0
0
0
0


hsa-miR-105
0
11
8
18
10
7
0


hsa-miR-105*
0
0
2
0
0
0
0


hsa-miR-106a
394
2073
2034
2465
2622
2258
829


hsa-miR-106a*
0
4
11
10
9
13
2


hsa-miR-106b
35123
3419
3847
4164
4238
3699
1679


hsa-miR-106b*
1664
114
108
154
119
137
136


hsa-miR-107
628
274
282
274
209
308
159


hsa-miR-10a
186
26956
24934
27899
28106
29258
17837


hsa-miR-10a*
0
115
112
129
148
136
123


hsa-miR-10b
7
40830
37049
43481
43238
44884
40079


hsa-miR-10b*
0
361
326
359
416
368
469


hsa-miR-1178
0
0
0
0
0
0
0


hsa-miR-1179
5
77
69
91
81
90
16


hsa-miR-1180
0
21
17
14
28
31
15


hsa-miR-1181
0
1
0
0
0
0
0


hsa-miR-1182
0
0
0
0
0
0
0


hsa-miR-1183
0
0
0
0
0
0
0


hsa-miR-1184
0
0
0
0
0
0
0


hsa-miR-1185
0
26
35
47
48
35
7


hsa-miR-1193
0
0
0
0
0
0
0


hsa-miR-1197
0
1
3
0
2
0
0


hsa-miR-1200
0
0
0
0
0
0
0


hsa-miR-1201
422
56
55
63
66
91
69


hsa-miR-1202
0
0
0
0
0
0
0


hsa-miR-1203
0
0
0
0
0
0
0


hsa-miR-1204
0
0
0
0
0
0
0


hsa-miR-1205
0
0
0
0
0
0
0


hsa-miR-1206
0
0
0
0
0
0
0


hsa-miR-1207-3p
0
0
0
0
0
0
0


hsa-miR-1207-5p
0
0
0
0
0
0
0


hsa-miR-1208
0
0
0
0
0
0
0


hsa-miR-122
4
5808
5666
6201
6163
6375
81


hsa-miR-122*
0
57
56
78
85
66
2


hsa-miR-1224-3p
0
0
0
1
0
0
0


hsa-miR-1224-5p
0
13
12
8
10
10
0


hsa-miR-1225-3p
0
1
0
0
0
0
0


hsa-miR-1225-5p
0
0
0
2
1
1
2


hsa-miR-1226
6
1
2
2
6
2
1


hsa-miR-1226*
7
0
0
3
0
0
0


hsa-miR-1227
0
0
0
0
0
0
0


hsa-miR-1228
0
1
1
0
1
2
2


hsa-miR-1228*
0
0
1
1
1
0
1


hsa-miR-1229
1
2
0
4
1
1
0


hsa-miR-1231
0
1
2
0
2
0
0


hsa-miR-1233
0
1
1
0
1
0
0


hsa-miR-1234
1
1
0
0
0
1
0


hsa-miR-1236
0
0
0
0
0
0
0


hsa-miR-1237
0
0
0
0
0
0
0


hsa-miR-1238
0
0
0
0
0
0
0


hsa-miR-124
0
1249
1154
1262
1285
1408
2


hsa-miR-124*
0
5
1
6
4
5
0


hsa-miR-1243
5
5
2
0
1
3
2


hsa-miR-1244
0
3
1
7
1
5
2


hsa-miR-1245
0
0
1
1
3
1
2


hsa-miR-1246
49235
5920
5881
6452
6358
6490
37368


hsa-miR-1247
0
92
92
112
120
93
46


hsa-miR-1248
2
28
22
22
19
16
27


hsa-miR-1249
1
25
26
31
32
29
11


hsa-miR-1250
0
4
1
1
0
2
0


hsa-miR-1251
0
110
82
133
128
137
0


hsa-miR-1252
0
1
2
1
2
0
0


hsa-miR-1253
0
0
0
0
0
0
0


hsa-miR-1254
34
1
3
12
7
8
2


hsa-miR-1255a
46
6
12
11
11
5
4


hsa-miR-1255b
6
3
3
11
2
7
3


hsa-miR-1256
5
0
4
1
0
0
0


hsa-miR-1257
27
0
0
0
0
0
0


hsa-miR-1258
0
2
3
1
4
1
2


hsa-miR-1259
268
38
29
29
30
43
69


hsa-miR-125a-3p
0
76
63
69
62
59
46


hsa-miR-125a-5p
3
14695
14391
16483
15940
16054
8971


hsa-miR-125b
738
30693
29479
33392
32497
33293
16929


hsa-miR-125b-1*
0
70
64
65
65
81
59


hsa-miR-125b-2*
421
1017
994
1074
1037
1170
640


hsa-miR-126
2
163374
163020
188443
191489
175101
137109


hsa-miR-126*
2
24037
23282
29250
28209
26553
25398


hsa-miR-1260
5
0
3
0
3
2
1


hsa-miR-1260b
60
14
16
15
11
12
8


hsa-miR-1261
0
0
0
0
0
0
0


hsa-miR-1262
0
12
7
11
9
17
12


hsa-miR-1263
0
0
0
0
0
0
0


hsa-miR-1264
0
0
1
2
1
2
0


hsa-miR-1265
0
3
0
0
0
0
0


hsa-miR-1266
0
5
3
3
4
0
8


hsa-miR-1267
0
0
0
0
1
0
0


hsa-miR-1268
504
20
22
30
24
20
9


hsa-miR-1269
0
14
15
13
14
13
0


hsa-miR-1270
474
66
47
48
67
74
37


hsa-miR-1271
0
50
48
58
58
40
39


hsa-miR-1272
0
0
0
0
0
1
0


hsa-miR-1273
0
0
0
0
0
0
0


hsa-miR-1273c
5
2
3
5
4
7
4


hsa-miR-1273d
3
0
0
1
0
0
0


hsa-miR-127-3p
3
1993
1787
2084
2026
2092
1242


hsa-miR-1274a
9
0
0
1
0
0
0


hsa-miR-1274b
40
64
61
70
72
49
23


hsa-miR-1275
192
25
27
36
23
30
16


hsa-miR-127-5p
0
343
290
391
386
362
69


hsa-miR-1276
23
1
0
0
2
1
0


hsa-miR-1277
79
39
22
37
26
32
4


hsa-miR-1278
21
1
1
1
1
2
0


hsa-miR-1279
0
0
0
0
0
0
0


hsa-miR-128
1231
2133
2052
2282
2117
2319
262


hsa-miR-1280
2
9
8
14
5
14
7


hsa-miR-1281
0
0
0
0
0
0
0


hsa-miR-1282
0
0
0
0
0
0
0


hsa-miR-1283
0
251
238
288
268
276
1


hsa-miR-1284
5
2
1
0
1
0
0


hsa-miR-1285
412
13
15
21
12
16
13


hsa-miR-1286
0
0
0
0
1
0
0


hsa-miR-1287
1
35
31
32
49
30
40


hsa-miR-1288
0
0
0
0
0
0
0


hsa-miR-1289
0
1
0
0
0
0
0


hsa-miR-129*
3
26
29
28
15
30
0


hsa-miR-1290
3
0
0
0
1
0
1


hsa-miR-1291
47
38
13
25
27
18
16


hsa-miR-1292
162
20
39
31
32
24
29


hsa-miR-1293
0
0
1
0
0
0
0


hsa-miR-129-3p
3
84
78
84
98
102
1


hsa-miR-1294
4
2
1
2
7
3
2


hsa-miR-1295
0
1
3
1
2
2
6


hsa-miR-129-5p
4
280
267
306
326
311
3


hsa-miR-1296
10
50
52
53
65
49
47


hsa-miR-1297
0
0
0
0
0
0
0


hsa-miR-1298
0
15
13
16
14
18
0


hsa-miR-1299
15
51
61
45
63
58
66


hsa-miR-1301
1
49
45
48
50
50
14


hsa-miR-1302
0
1
0
0
0
0
0


hsa-miR-1303
27
7
5
7
8
17
7


hsa-miR-1304
7
3
1
3
2
3
0


hsa-miR-1305
0
2
8
6
7
5
5


hsa-miR-1306
1
0
3
2
3
1
5


hsa-miR-1307
1860
328
292
362
324
337
196


hsa-miR-1308
722
471
506
521
567
519
206


hsa-miR-130a
4
2674
2388
2934
2721
2777
2772


hsa-miR-130a*
0
5
2
6
8
6
13


hsa-miR-130b
3376
456
434
625
526
455
217


hsa-miR-130b*
81
85
63
66
63
72
21


hsa-miR-132
77
750
687
834
904
797
686


hsa-miR-132*
6
125
110
117
116
119
57


hsa-miR-1321
0
0
0
0
0
1
0


hsa-miR-1322
4
1
2
0
0
2
0


hsa-miR-1323
0
3503
3317
2994
2327
3903
8


hsa-miR-1324
0
0
0
0
0
0
0


hsa-miR-133a
2
18167
18669
22360
22448
19771
51


hsa-miR-133b
0
556
566
637
624
553
2


hsa-miR-134
0
172
171
185
212
192
200


hsa-miR-135a
1
9461
8663
9893
7606
10424
105


hsa-miR-135a*
0
6
6
4
8
9
0


hsa-miR-135b
0
2934
2829
3189
2875
3219
357


hsa-miR-135b*
0
20
11
18
17
19
7


hsa-miR-136
0
2340
1609
1993
1920
2381
500


hsa-miR-136*
1
413
380
489
489
467
170


hsa-miR-137
0
509
472
442
444
548
7


hsa-miR-138
3027
968
858
1165
1119
984
67


hsa-miR-138-1*
58
27
26
26
27
27
8


hsa-miR-138-2*
0
8
4
6
5
4
0


hsa-miR-139-3p
9
125
130
105
116
131
163


hsa-miR-139-5p
11
1451
1370
1583
1571
1574
1683


hsa-miR-140-3p
67006
37936
34949
36311
36598
41136
16014


hsa-miR-140-5p
3672
6850
6579
8128
7884
7500
5245


hsa-miR-141
79
6530
6644
6908
7685
7532
3759


hsa-miR-141*
0
155
128
159
131
134
152


hsa-miR-142-3p
169524
71892
68818
82272
79112
77431
8578


hsa-miR-142-5p
16201
2014
1877
2148
2101
2323
882


hsa-miR-143
17640
1884521
1792761
2010306
2107243
2088207
1074889


hsa-miR-143*
11
14298
14952
19113
18944
15393
4904


hsa-miR-144
0
1335
1046
1285
1271
1455
132


hsa-miR-144*
13
7752
7376
8357
8770
8522
3235


hsa-miR-145
40
64408
62487
79740
74320
68219
26847


hsa-miR-145*
3
2495
2415
2993
2747
2567
1465


hsa-miR-1468
1
4
5
4
8
7
1


hsa-miR-1469
0
0
0
0
0
0
0


hsa-miR-146a
45680
8759
8469
9841
9877
9557
9883


hsa-miR-146a*
0
2
0
4
1
3
4


hsa-miR-146b-3p
40
20
10
23
25
23
46


hsa-miR-146b-5p
51686
64507
64864
73443
75492
70909
105601


hsa-miR-147
0
0
0
0
0
0
0


hsa-miR-1470
0
0
0
0
0
0
0


hsa-miR-1471
0
0
0
0
0
0
0


hsa-miR-147b
0
5
8
9
7
4
13


hsa-miR-148a
167654
83936
69915
67362
59455
92561
55496


hsa-miR-148a*
2741
231
173
236
232
238
257


hsa-miR-148b
18296
12418
10163
9757
8603
13625
4727


hsa-miR-148b*
2605
303
339
352
366
365
204


hsa-miR-149
22
198
187
269
258
224
126


hsa-miR-149*
0
2
2
1
1
4
2


hsa-miR-150
1
2028
1981
2312
2285
2182
1767


hsa-miR-150*
0
17
26
37
30
19
6


hsa-miR-151-3p
4140
9265
8272
9478
9542
9887
6249


hsa-miR-151-5p
1069
9893
9391
10906
10349
10836
6486


hsa-miR-152
881
12737
10667
10744
9523
13596
9459


hsa-miR-153
5
314
247
325
302
316
54


hsa-miR-1537
0
3
1
2
3
3
0


hsa-miR-1538
0
1
1
0
0
0
0


hsa-miR-1539
0
0
0
0
0
0
0


hsa-miR-154
0
65
59
72
72
93
24


hsa-miR-154*
0
32
27
17
28
20
4


hsa-miR-155
118345
3723
3704
4428
4788
4169
2397


hsa-miR-155*
477
0
1
1
2
0
0


hsa-miR-15a
11248
2927
2588
3145
3253
2998
1562


hsa-miR-15a*
8
3
3
4
1
6
2


hsa-miR-15b
4272
1346
1127
1151
1156
1456
1012


hsa-miR-15b*
712
92
78
100
95
101
50


hsa-miR-16
25236
33304
35260
36274
35048
35434
22850


hsa-miR-16-1*
242
6
12
9
4
6
2


hsa-miR-16-2*
277
81
97
115
90
77
92


hsa-miR-17
75571
8032
7314
9397
9426
8462
3575


hsa-miR-17*
7638
396
347
387
360
469
69


hsa-miR-181a
14796
21358
19352
24301
24133
24053
7752


hsa-miR-181a*
977
244
223
282
287
246
53


hsa-miR-181a-2*
4
267
221
301
249
267
96


hsa-miR-181b
3623
2995
2745
3337
3228
3420
1302


hsa-miR-181c
15
594
605
682
662
698
314


hsa-miR-181c*
4
64
78
75
83
95
45


hsa-miR-181d
16
333
312
372
358
373
231


hsa-miR-182
17542
4447
3961
4784
4611
4786
20534


hsa-miR-182*
0
3
0
0
0
0
1


hsa-miR-1825
0
0
0
0
0
0
0


hsa-miR-1826
201
209
183
222
226
236
145


hsa-miR-1827
0
0
0
0
1
0
0


hsa-miR-183
4237
1068
920
1093
972
1165
3294


hsa-miR-183*
48
3
2
3
4
1
8


hsa-miR-184
178
266
245
274
292
312
47


hsa-miR-185
1420
917
965
1062
1034
1015
640


hsa-miR-185*
21
15
14
11
8
17
8


hsa-miR-186
23895
10864
11534
15131
14516
11876
9468


hsa-miR-186*
242
18
13
13
12
18
7


hsa-miR-187
0
61
66
77
62
76
24


hsa-miR-187*
0
6
4
9
4
4
0


hsa-miR-188-3p
0
5
4
4
1
3
5


hsa-miR-188-5p
0
66
48
61
48
71
46


hsa-miR-18a
9458
450
481
506
575
498
132


hsa-miR-18a*
105
16
15
22
10
8
14


hsa-miR-18b
14
17
10
12
12
21
4


hsa-miR-18b*
0
3
2
1
2
0
0


hsa-miR-190
682
1085
1022
1248
1277
1256
529


hsa-miR-1908
0
1
0
0
1
4
0


hsa-miR-1909
0
0
0
0
0
0
0


hsa-miR-1909*
0
0
0
0
0
0
0


hsa-miR-190b
3
17
16
28
30
22
258


hsa-miR-191
49626
21938
20938
23858
21723
24104
14960


hsa-miR-191*
85
7
9
7
8
10
3


hsa-miR-1910
0
0
0
0
1
0
0


hsa-miR-1911
0
12
11
3
13
8
0


hsa-miR-1911*
0
0
0
0
0
0
0


hsa-miR-1912
0
3
1
5
3
3
0


hsa-miR-1913
0
0
0
0
1
0
0


hsa-miR-1914
0
0
0
0
0
0
1


hsa-miR-1914*
0
0
1
0
0
0
0


hsa-miR-1915
0
0
0
0
0
1
0


hsa-miR-1915*
0
0
0
0
0
0
0


hsa-miR-192
12660
67252
64208
78702
77501
72613
1512


hsa-miR-192*
13
108
96
114
116
86
9


hsa-miR-193a-3p
2
321
195
260
226
300
597


hsa-miR-193a-5p
0
274
240
270
305
250
1038


hsa-miR-193b
3731
1819
1714
2035
1909
1997
3930


hsa-miR-193b*
387
53
61
65
56
69
131


hsa-miR-194
1023
29917
30929
37348
37266
32599
483


hsa-miR-194*
23
46
41
43
42
49
1


hsa-miR-195
1
5404
5286
4950
4646
5711
8702


hsa-miR-195*
0
24
26
30
34
35
41


hsa-miR-196a
59
559
574
687
745
676
623


hsa-miR-196a*
3
1
0
3
2
1
1


hsa-miR-196b
0
1600
1451
1723
1821
1707
742


hsa-miR-196b*
0
4
2
3
0
5
2


hsa-miR-197
322
1102
1084
1283
1209
1109
865


hsa-miR-1972
0
0
0
0
0
0
0


hsa-miR-1973
9
9
8
5
6
10
4


hsa-miR-1975
1004
3292
3615
4209
4391
3345
9392


hsa-miR-1976
10
10
4
12
4
6
4


hsa-miR-1979
1720
1998
1856
2198
2246
2083
14700


hsa-miR-198
0
0
0
0
0
0
1


hsa-miR-199a-3p
5
20978
20327
25304
24599
23143
29779


hsa-miR-199a-5p
1
3977
3908
4604
4598
4225
5084


hsa-miR-199b-3p
5
20978
20327
25304
24599
23143
29779


hsa-miR-199b-5p
0
957
864
1106
1119
1025
2982


hsa-miR-19a
5598
2533
2848
3908
3726
2702
903


hsa-miR-19a*
18
0
1
0
1
1
0


hsa-miR-19b
18432
9896
10962
14056
13647
10888
9189


hsa-miR-19b-1*
256
25
22
23
20
21
13


hsa-miR-19b-2*
0
0
0
0
0
0
0


hsa-miR-200a
5
2454
2278
2445
2540
2705
1661


hsa-miR-200a*
0
44
53
54
49
71
47


hsa-miR-200b
2
3797
3888
4427
4654
4284
5020


hsa-miR-200b*
0
410
390
415
381
413
423


hsa-miR-200c
155
30724
29255
31830
33480
34310
43316


hsa-miR-200c*
2
7
11
11
12
11
6


hsa-miR-202
0
366
321
280
191
397
1


hsa-miR-202*
1
7203
6848
8579
8374
8087
86


hsa-miR-203
0
4340
3971
4752
4447
4863
1179


hsa-miR-204
0
689
695
838
784
810
67


hsa-miR-205
0
5042
4656
5518
5108
5408
55620


hsa-miR-205*
0
2
1
3
1
0
4


hsa-miR-2052
0
0
0
0
0
0
0


hsa-miR-2053
0
0
0
0
0
0
0


hsa-miR-2054
0
0
0
0
0
0
0


hsa-miR-206
0
13453
13591
15087
16300
15006
4


hsa-miR-208a
0
64
34
54
43
66
0


hsa-miR-208b
0
322
319
343
304
354
0


hsa-miR-20a
138720
9808
9528
10260
10813
10890
5182


hsa-miR-20a*
454
419
476
560
521
420
168


hsa-miR-20b
2
1344
1204
1591
1628
1459
138


hsa-miR-20b*
0
20
13
18
20
23
3


hsa-miR-21
1852326
331847
313309
353783
377299
368479
300625


hsa-miR-21*
2992
117
104
123
126
122
102


hsa-miR-210
5008
992
971
1242
1326
974
254


hsa-miR-211
0
2
1
4
0
3
2


hsa-miR-2110
29
93
89
60
36
105
53


hsa-miR-2113
0
0
0
0
0
0
0


hsa-miR-2114
0
5
7
6
5
8
1


hsa-miR-2114*
0
0
1
1
0
0
0


hsa-miR-2115
0
3
2
1
3
2
26


hsa-miR-2115*
0
1
7
2
1
2
40


hsa-miR-2116
1
3
4
4
5
0
1


hsa-miR-2116*
0
2
0
3
3
3
3


hsa-miR-2117
0
0
0
0
0
0
0


hsa-miR-212
0
25
23
25
28
24
19


hsa-miR-214
0
916
830
776
685
984
1207


hsa-miR-214*
0
167
154
183
207
209
129


hsa-miR-215
142
51160
49148
56830
57666
56128
414


hsa-miR-216a
2
20
18
25
20
18
7


hsa-miR-216b
0
70
63
76
87
74
12


hsa-miR-217
7
62
47
61
54
66
20


hsa-miR-218
1
2040
2073
2559
2571
2186
1055


hsa-miR-218-1*
0
56
48
63
49
58
58


hsa-miR-218-2*
0
0
3
2
1
4
0


hsa-miR-219-1-
71
2
2
1
2
6
3


3p









hsa-miR-219-2-
0
902
1342
1709
1858
2121
2


3p









hsa-miR-219-5p
30
44
42
53
39
41
1


hsa-miR-22
926
12597
11782
13008
13488
13549
11463


hsa-miR-22*
53
866
967
1293
1130
948
1006


hsa-miR-220a
0
0
0
0
0
0
0


hsa-miR-220b
0
0
0
0
0
0
0


hsa-miR-220c
0
0
0
0
0
0
0


hsa-miR-221
285
3056
3079
3686
3758
3422
2113


hsa-miR-221*
391
1229
999
1015
861
1312
389


hsa-miR-222
213
7470
7604
9085
8803
8291
5395


hsa-miR-222*
75
38
25
39
23
33
10


hsa-miR-223
3
2838
2663
3045
3179
3067
1895


hsa-miR-223*
1
94
117
201
183
101
51


hsa-miR-224
0
587
531
684
703
650
997


hsa-miR-224*
0
28
30
30
26
34
77


hsa-miR-2276
22
6
4
6
4
8
3


hsa-miR-2277
1
4
0
6
1
4
2


hsa-miR-2278
0
0
0
1
2
0
4


hsa-miR-2355
0
5
8
9
4
3
7


hsa-miR-23a
360
15888
14598
17234
16789
17131
21956


hsa-miR-23a*
3
4
3
0
0
1
3


hsa-miR-23b
474
14755
13404
15889
15851
15682
5726


hsa-miR-23b*
6
21
18
17
13
16
6


hsa-miR-24
1815
116792
122743
140600
135084
126140
67259


hsa-miR-24-1*
8
276
284
347
304
343
56


hsa-miR-24-2*
14
584
523
594
525
558
272


hsa-miR-25
7496
1904
1720
1980
1914
2048
1898


hsa-miR-25*
61
30
29
23
10
38
11


hsa-miR-26a
23823
139238
120963
131441
135641
156484
139950


hsa-miR-26a-1*
0
4
7
5
1
6
4


hsa-miR-26a-2*
58
68
51
77
70
59
63


hsa-miR-26b
11315
37731
28997
31628
32208
42694
29712


hsa-miR-26b*
43
50
54
56
63
61
59


hsa-miR-27a
362
19157
19019
21101
22219
21482
21448


hsa-miR-27a*
20
43
42
73
61
49
36


hsa-miR-27b
2465
47617
43110
45439
47218
52830
16131


hsa-miR-27b*
135
633
790
963
965
725
207


hsa-miR-28-3p
1396
3372
2921
3435
3485
3521
2162


hsa-miR-28-5p
1019
3040
2861
3393
3391
3514
3027


hsa-miR-2861
0
0
0
0
0
0
0


hsa-miR-2909
0
0
0
0
0
0
0


hsa-miR-296-3p
50
4
3
4
5
4
10


hsa-miR-296-5p
79
32
33
46
46
30
57


hsa-miR-297
0
0
0
0
0
0
0


hsa-miR-298
0
0
0
0
0
0
0


hsa-miR-299-3p
0
33
31
32
43
40
18


hsa-miR-299-5p
0
72
78
91
88
71
34


hsa-miR-29a
40124
61875
60909
79815
80527
67864
31795


hsa-miR-29a*
991
294
301
347
369
332
167


hsa-miR-29b
14193
26166
18024
24954
24981
29084
5148


hsa-miR-29b-1*
120
16
18
28
32
19
9


hsa-miR-29b-2*
147
146
115
155
155
131
87


hsa-miR-29c
1880
3927
3550
4318
4137
4173
3491


hsa-miR-29c*
162
359
397
441
421
380
317


hsa-miR-300
0
0
0
0
0
0
0


hsa-miR-301a
421
98
82
92
93
120
10


hsa-miR-301b
169
14
13
8
16
5
1


hsa-miR-302a
0
2
3
3
2
10
0


hsa-miR-302a*
0
12
5
13
10
12
0


hsa-miR-302b
0
4
6
14
9
9
0


hsa-miR-302b*
0
0
0
0
0
2
0


hsa-miR-302c
0
2
2
1
1
1
0


hsa-miR-302c*
0
0
0
0
0
0
0


hsa-miR-302d
0
3
1
1
1
4
0


hsa-miR-302d*
0
0
0
0
0
0
0


hsa-miR-302e
0
0
0
0
0
0
0


hsa-miR-302f
0
0
0
0
0
0
0


hsa-miR-3065-3p
1
112
102
122
100
116
35


hsa-miR-3065-5p
0
114
114
122
119
110
22


hsa-miR-3074
1
1
0
1
1
2
1


hsa-miR-30a
425
134091
121854
147246
139908
146572
158595


hsa-miR-30a*
4
5542
4869
5972
5456
5900
8890


hsa-miR-30b
7820
25303
23222
30378
31188
28066
16765


hsa-miR-30b*
87
46
53
77
76
50
39


hsa-miR-30c
13867
33356
32048
37747
37655
35877
24196


hsa-miR-30c-1*
54
28
31
50
51
25
26


hsa-miR-30c-2*
0
367
324
335
330
411
491


hsa-miR-30d
235117
66258
63436
72267
71041
71729
54966


hsa-miR-30d*
450
83
84
90
88
83
74


hsa-miR-30e
238220
47135
46880
52861
55314
52193
14558


hsa-miR-30e*
11783
6097
5381
6546
6164
6665
3187


hsa-miR-31
0
776
768
823
692
794
654


hsa-miR-31*
0
22
31
21
26
31
40


hsa-miR-3115
2
3
1
2
1
2
2


hsa-miR-3116
0
0
1
0
2
0
0


hsa-miR-3117
0
10
7
8
6
6
0


hsa-miR-3118
0
0
0
0
0
0
0


hsa-miR-3119
0
0
0
0
0
0
0


hsa-miR-3120
1
0
0
1
0
2
0


hsa-miR-3121
4
1
0
0
0
1
1


hsa-miR-3122
2
0
0
0
0
0
1


hsa-miR-3123
0
0
0
0
0
0
0


hsa-miR-3124
0
3
3
1
3
3
2


hsa-miR-3125
0
2
3
0
1
4
1


hsa-miR-3126-3p
0
0
0
2
0
0
0


hsa-miR-3126-5p
0
1
0
0
0
3
0


hsa-miR-3127
15
5
1
2
4
0
1


hsa-miR-3128
13
0
4
0
0
4
0


hsa-miR-3129
0
5
3
8
5
7
14


hsa-miR-3130-3p
0
0
0
0
0
0
0


hsa-miR-3130-5p
7
2
1
0
1
1
1


hsa-miR-3131
0
0
0
0
0
1
0


hsa-miR-3132
0
1
0
0
1
0
0


hsa-miR-3133
5
1
1
2
1
0
2


hsa-miR-3134
3
1
1
1
0
3
1


hsa-miR-3135
0
0
0
0
0
0
0


hsa-miR-3136
8
2
2
0
0
0
0


hsa-miR-3137
0
0
0
0
0
0
0


hsa-miR-3138
1
5
3
4
1
0
2


hsa-miR-3139
1
1
0
0
3
0
0


hsa-miR-3140
6
2
1
0
3
0
1


hsa-miR-3141
1
1
1
1
0
0
0


hsa-miR-3142
0
0
0
0
0
0
0


hsa-miR-3143
1
1
0
1
1
0
0


hsa-miR-3144-3p
0
0
0
1
0
1
1


hsa-miR-3144-5p
0
0
0
0
0
2
1


hsa-miR-3145
0
0
0
0
3
2
0


hsa-miR-3146
10
3
1
2
2
6
1


hsa-miR-3147
0
0
0
0
0
0
0


hsa-miR-3148
0
0
0
0
0
1
0


hsa-miR-3149
0
1
2
1
1
2
0


hsa-miR-3150
0
1
0
0
0
0
0


hsa-miR-3151
0
0
0
0
0
0
0


hsa-miR-3152
0
0
0
0
0
0
0


hsa-miR-3153
0
0
0
0
0
0
0


hsa-miR-3154
0
7
9
13
5
13
2


hsa-miR-3155
1
0
0
1
0
0
0


hsa-miR-3156
0
0
0
0
0
1
0


hsa-miR-3157
1
1
1
1
1
2
2


hsa-miR-3158
135
3
6
7
10
8
1


hsa-miR-3159
0
0
0
0
0
0
0


hsa-miR-3160
0
0
1
0
0
0
0


hsa-miR-3161
6
0
1
1
0
3
0


hsa-miR-3162
0
1
0
0
0
0
0


hsa-miR-3163
0
1
1
1
1
1
1


hsa-miR-3164
11
3
3
3
7
6
2


hsa-miR-3165
0
0
0
0
0
0
0


hsa-miR-3166
0
0
0
0
0
0
1


hsa-miR-3167
0
2
0
1
1
0
0


hsa-miR-3168
0
0
0
0
0
0
2


hsa-miR-3169
0
0
0
0
0
0
0


hsa-miR-3170
0
0
1
0
0
0
0


hsa-miR-3171
0
0
1
0
0
1
0


hsa-miR-3172
22
49
37
44
37
47
188


hsa-miR-3173
2
0
0
0
0
0
1


hsa-miR-3174
10
1
1
0
0
0
1


hsa-miR-3175
0
0
0
0
2
0
1


hsa-miR-3176
14
2
1
5
7
4
1


hsa-miR-3177
9
3
5
5
4
5
0


hsa-miR-3178
0
0
0
0
0
0
0


hsa-miR-3179
3
2
0
0
0
1
1


hsa-miR-3180-3p
7
1
0
2
0
0
1


hsa-miR-3180-5p
0
0
1
0
2
0
0


hsa-miR-3181
2
0
0
1
0
1
0


hsa-miR-3182
0
0
0
0
0
0
0


hsa-miR-3183
0
0
0
0
0
0
0


hsa-miR-3184
2
0
0
0
0
0
0


hsa-miR-3185
0
0
0
0
0
0
0


hsa-miR-3186-3p
0
0
0
0
0
0
0


hsa-miR-3186-5p
0
0
0
0
0
1
0


hsa-miR-3187
6
3
1
3
0
3
0


hsa-miR-3188
6
8
10
13
9
7
2


hsa-miR-3189
0
0
0
2
0
0
0


hsa-miR-3190-3p
3
1
0
1
1
2
1


hsa-miR-3190-5p
3
1
0
1
1
2
1


hsa-miR-3191
2
0
0
0
0
0
0


hsa-miR-3192
1
1
1
2
1
0
2


hsa-miR-3193
0
0
0
0
0
1
1


hsa-miR-3194
10
3
2
5
2
2
5


hsa-miR-3195
0
1
0
0
0
1
2


hsa-miR-3196
0
2
1
0
3
0
2


hsa-miR-3197
0
0
0
0
0
0
0


hsa-miR-3198
0
0
0
0
0
1
0


hsa-miR-3199
0
6
6
3
2
5
9


hsa-miR-32
5470
2098
1462
1838
1996
2306
1090


hsa-miR-32*
68
26
24
24
29
11
13


hsa-miR-3200
1
37
39
52
34
51
6


hsa-miR-3201
0
0
0
0
0
0
0


hsa-miR-3202
0
1
0
0
1
0
0


hsa-miR-320a
1244
6971
7889
7436
7785
7380
9423


hsa-miR-320b
51
417
465
390
409
449
2305


hsa-miR-320c
34
281
247
266
256
296
1932


hsa-miR-320d
46
314
284
321
258
359
2160


hsa-miR-320e
6
9
10
13
4
9
58


hsa-miR-323-3p
0
51
42
56
46
45
11


hsa-miR-323-5p
0
0
2
0
1
0
0


hsa-miR-323b-3p
0
22
12
24
24
24
1


hsa-miR-323b-5p
0
0
1
0
0
0
1


hsa-miR-324-3p
81
88
92
92
102
95
109


hsa-miR-324-5p
271
177
187
189
174
183
127


hsa-miR-325
0
0
0
0
0
0
0


hsa-miR-326
0
21
21
19
20
16
16


hsa-miR-328
10
234
256
318
319
224
99


hsa-miR-329
0
130
98
100
107
108
43


hsa-miR-330-3p
255
263
217
154
144
284
46


hsa-miR-330-5p
299
46
33
32
42
44
13


hsa-miR-331-3p
281
134
178
143
156
135
88


hsa-miR-331-5p
223
30
24
38
47
26
25


hsa-miR-335
0
2630
2223
2454
2057
2932
4406


hsa-miR-335*
1
533
585
687
626
570
505


hsa-miR-337-3p
0
323
326
405
308
339
200


hsa-miR-337-5p
0
270
247
249
226
301
111


hsa-miR-338-3p
0
707
633
705
767
764
49


hsa-miR-338-5p
0
341
359
464
420
420
23


hsa-miR-339-3p
2226
606
610
725
721
707
442


hsa-miR-339-5p
413
186
163
178
203
206
211


hsa-miR-33a
1634
753
748
945
796
830
95


hsa-miR-33a*
223
15
12
16
16
14
9


hsa-miR-33b
385
46
47
67
59
52
58


hsa-miR-33b*
46
2
1
3
3
3
2


hsa-miR-340
0
1628
1439
1189
1024
1805
891


hsa-miR-340*
0
170
162
157
184
161
38


hsa-miR-342-3p
1703
1869
1713
1975
2067
1940
2239


hsa-miR-342-5p
185
148
166
164
193
170
329


hsa-miR-345
4
269
289
274
273
276
235


hsa-miR-346
0
9
12
12
13
4
3


hsa-miR-34a
0
1016
1070
1335
1289
1090
1240


hsa-miR-34a*
0
10
6
8
6
10
9


hsa-miR-34b
0
93
81
100
104
99
9


hsa-miR-34b*
0
84
103
103
118
118
6


hsa-miR-34c-3p
0
128
137
153
174
151
17


hsa-miR-34c-5p
1
2391
2526
2797
2763
2601
217


hsa-miR-361-3p
300
272
276
340
317
287
269


hsa-miR-361-5p
600
1507
1558
1786
1855
1606
1375


hsa-miR-362-3p
0
49
48
64
65
58
16


hsa-miR-362-5p
0
193
164
182
181
185
43


hsa-miR-363
0
1302
1223
1382
1458
1472
157


hsa-miR-363*
0
0
1
1
3
3
1


hsa-miR-365
2549
1375
1221
1388
1441
1463
2373


hsa-miR-365*
0
10
10
6
17
13
62


hsa-miR-367
0
1
2
3
2
1
0


hsa-miR-367*
0
0
0
0
0
0
0


hsa-miR-369-3p
9
577
532
764
701
636
208


hsa-miR-369-5p
0
100
98
130
119
114
68


hsa-miR-370
0
61
63
54
60
50
7


hsa-miR-371-3p
0
3
1
0
3
2
0


hsa-miR-371-5p
0
10
13
12
15
10
0


hsa-miR-372
0
34
33
39
40
35
0


hsa-miR-373
0
33
20
41
20
22
0


hsa-miR-373*
0
2
0
3
1
3
0


hsa-miR-374a
1642
4464
5594
8500
9392
5156
2047


hsa-miR-374a*
23689
5542
5001
6338
6354
6298
3692


hsa-miR-374b
543
3517
2589
3518
3701
4095
3002


hsa-miR-374b*
154
105
87
105
123
120
35


hsa-miR-375
1
1319
1356
1703
1480
1624
638


hsa-miR-376a
0
87
66
76
89
73
27


hsa-miR-376a*
0
128
121
166
130
146
53


hsa-miR-376b
0
103
82
114
112
105
25


hsa-miR-376c
0
1684
1503
1867
1742
1787
643


hsa-miR-377
0
558
361
555
569
632
122


hsa-miR-377*
0
2
3
8
6
6
4


hsa-miR-378
1201524
154120
139944
167798
163364
166366
118921


hsa-miR-378*
219
224
214
319
297
265
163


hsa-miR-378b
3
2
4
4
7
4
4


hsa-miR-378c
15826
1643
1586
1821
1742
1796
1200


hsa-miR-379
1
3016
2768
3255
3392
3458
1874


hsa-miR-379*
0
23
16
26
20
15
4


hsa-miR-380
0
14
7
20
7
10
5


hsa-miR-380*
0
30
21
24
22
25
4


hsa-miR-381
0
1429
1322
1397
1482
1589
443


hsa-miR-382
0
135
142
129
154
159
79


hsa-miR-383
0
55
74
56
64
79
29


hsa-miR-384
0
0
0
0
0
0
0


hsa-miR-409-3p
0
92
99
143
114
123
57


hsa-miR-409-5p
0
27
29
57
36
36
16


hsa-miR-410
0
94
102
105
91
118
15


hsa-miR-411
2
823
809
981
1042
911
356


hsa-miR-411*
0
59
45
40
36
48
10


hsa-miR-412
0
0
0
0
1
0
0


hsa-miR-421
48
185
154
135
135
185
131


hsa-miR-422a
7
1
0
0
0
1
0


hsa-miR-423-3p
2241
2289
2145
2407
2539
2593
1694


hsa-miR-423-5p
11168
4665
3912
3296
2517
5015
4426


hsa-miR-424
1
17513
16557
18944
17115
18702
4393


hsa-miR-424*
0
166
152
174
159
181
27


hsa-miR-425
20272
5387
4390
5330
5548
5778
1961


hsa-miR-425*
463
181
166
241
248
219
224


hsa-miR-4251
0
0
0
0
0
0
0


hsa-miR-4252
0
0
0
0
0
0
0


hsa-miR-4253
0
0
0
0
0
0
0


hsa-miR-4254
0
0
1
0
0
1
0


hsa-miR-4255
0
0
0
0
0
0
0


hsa-miR-4256
0
0
0
0
0
0
0


hsa-miR-4257
0
0
0
0
0
0
0


hsa-miR-4258
0
0
0
0
0
0
0


hsa-miR-4259
0
0
0
0
0
0
0


hsa-miR-4260
0
0
0
0
0
0
0


hsa-miR-4261
0
0
0
0
0
0
0


hsa-miR-4262
0
0
0
0
0
0
0


hsa-miR-4263
0
0
0
0
0
0
0


hsa-miR-4264
0
0
0
0
0
0
0


hsa-miR-4265
0
0
0
0
0
0
0


hsa-miR-4266
0
0
0
0
0
0
0


hsa-miR-4267
0
0
0
0
0
0
0


hsa-miR-4268
0
0
0
0
0
0
0


hsa-miR-4269
0
0
0
0
0
0
0


hsa-miR-4270
0
0
0
0
0
0
0


hsa-miR-4271
0
0
0
0
0
0
0


hsa-miR-4272
0
0
0
0
0
0
0


hsa-miR-4273
0
0
0
0
0
0
0


hsa-miR-4274
0
0
0
0
0
0
0


hsa-miR-4275
0
0
0
0
0
0
0


hsa-miR-4276
0
0
0
0
0
0
0


hsa-miR-4277
0
0
0
0
0
0
0


hsa-miR-4278
0
0
0
0
0
0
0


hsa-miR-4279
0
0
0
0
0
0
0


hsa-miR-4280
0
0
0
0
0
0
0


hsa-miR-4281
0
0
0
0
0
0
0


hsa-miR-4282
0
0
0
0
0
0
0


hsa-miR-4283
0
0
0
0
0
0
0


hsa-miR-4284
38
97
85
83
79
110
311


hsa-miR-4285
0
0
0
0
0
0
0


hsa-miR-4286
63
95
91
111
101
93
131


hsa-miR-4287
0
0
0
0
0
0
0


hsa-miR-4288
0
0
0
0
0
0
0


hsa-miR-4289
0
0
0
0
0
0
0


hsa-miR-429
0
1694
1868
2088
2178
1959
1384


hsa-miR-4290
0
0
0
0
0
0
0


hsa-miR-4291
0
0
0
0
0
0
0


hsa-miR-4292
0
0
0
0
0
0
0


hsa-miR-4293
0
0
0
0
0
0
0


hsa-miR-4294
0
0
0
0
0
0
0


hsa-miR-4295
0
0
0
0
0
0
0


hsa-miR-4296
0
0
0
0
1
0
0


hsa-miR-4297
0
0
0
0
0
0
0


hsa-miR-4298
0
0
0
0
0
0
0


hsa-miR-4299
0
0
0
0
0
0
0


hsa-miR-4300
0
0
0
0
0
0
0


hsa-miR-4301
0
0
0
0
0
0
0


hsa-miR-4302
0
0
0
0
0
0
0


hsa-miR-4303
0
0
0
0
0
0
0


hsa-miR-4304
0
0
0
0
0
0
0


hsa-miR-4305
0
0
0
0
0
0
0


hsa-miR-4306
0
0
0
0
0
0
0


hsa-miR-4307
0
0
0
0
0
0
0


hsa-miR-4308
0
0
0
0
0
0
0


hsa-miR-4309
0
0
0
0
0
0
0


hsa-miR-431
0
21
9
10
8
11
2


hsa-miR-431*
0
13
11
8
11
12
5


hsa-miR-4310
0
0
0
0
0
0
0


hsa-miR-4311
0
0
0
0
0
0
0


hsa-miR-4312
0
0
0
0
0
0
0


hsa-miR-4313
0
0
0
0
0
0
0


hsa-miR-4314
0
0
0
0
0
0
0


hsa-miR-4315
0
0
0
0
0
0
0


hsa-miR-4316
0
0
0
0
0
0
0


hsa-miR-4317
0
0
0
0
0
0
0


hsa-miR-4318
0
0
0
0
0
0
0


hsa-miR-4319
0
0
0
0
0
0
0


hsa-miR-432
0
74
79
71
84
92
71


hsa-miR-432*
0
2
1
0
2
0
0


hsa-miR-4320
0
0
0
0
0
0
0


hsa-miR-4321
0
0
0
0
0
0
0


hsa-miR-4322
0
0
0
0
0
0
0


hsa-miR-4323
0
0
0
0
0
1
0


hsa-miR-4324
0
0
0
0
0
0
0


hsa-miR-4325
0
0
0
0
0
0
0


hsa-miR-4326
24
4
6
3
5
5
2


hsa-miR-4327
0
0
0
0
0
0
0


hsa-miR-4328
0
2
7
7
3
6
0


hsa-miR-4329
0
0
0
0
0
0
0


hsa-miR-433
0
50
32
38
38
45
10


hsa-miR-4330
0
0
0
0
0
0
0


hsa-miR-448
0
1
1
0
0
1
0


hsa-miR-449a
10
679
644
797
884
758
13


hsa-miR-449b
2
108
94
129
111
149
5


hsa-miR-449b*
0
5
1
1
4
2
0


hsa-miR-449c
5
110
102
101
101
134
2


hsa-miR-449c*
0
0
0
0
0
0
0


hsa-miR-450a
0
876
877
1040
1138
927
261


hsa-miR-450b-3p
0
3
4
0
8
3
2


hsa-miR-450b-5p
0
385
341
414
431
420
101


hsa-miR-451
5
54380
51253
59248
61747
60268
25583


hsa-miR-452
0
6482
5791
6960
6629
7039
19691


hsa-miR-452*
0
136
149
150
160
147
389


hsa-miR-454
939
1874
1335
1717
1577
2044
615


hsa-miR-454*
6
5
8
14
7
8
3


hsa-miR-455-3p
0
1300
1114
1459
1285
1398
456


hsa-miR-455-5p
0
311
241
319
326
303
131


hsa-miR-466
0
0
0
1
0
0
0


hsa-miR-483-3p
0
31
40
31
27
36
11


hsa-miR-483-5p
0
220
208
223
160
229
156


hsa-miR-484
367
204
170
193
238
222
116


hsa-miR-485-3p
0
47
48
40
41
34
7


hsa-miR-485-5p
0
75
59
61
41
74
18


hsa-miR-486-3p
6
25
14
28
20
38
5


hsa-miR-486-5p
52
1057
1088
1112
1126
1079
435


hsa-miR-487a
0
43
35
55
40
57
14


hsa-miR-487b
3
213
209
231
266
225
56


hsa-miR-488
0
21
28
23
40
24
10


hsa-miR-488*
0
0
0
3
4
2
0


hsa-miR-489
0
54
46
29
35
45
46


hsa-miR-490-3p
0
89
93
107
99
105
0


hsa-miR-490-5p
0
100
74
97
90
82
1


hsa-miR-491-3p
19
3
8
4
7
7
1


hsa-miR-491-5p
22
53
48
65
37
61
65


hsa-miR-492
0
0
0
3
0
1
0


hsa-miR-493
0
82
80
75
104
94
10


hsa-miR-493*
0
201
190
248
207
198
23


hsa-miR-494
4
212
200
227
210
196
167


hsa-miR-495
0
197
201
231
248
267
62


hsa-miR-496
0
6
5
7
8
12
1


hsa-miR-497
1
737
729
811
815
831
1169


hsa-miR-497*
0
21
25
42
38
26
6


hsa-miR-498
0
105
68
88
91
94
0


hsa-miR-499-3p
2
600
758
911
1002
652
12


hsa-miR-499-5p
7
1417
1293
1309
1223
1611
27


hsa-miR-500
0
73
86
101
117
107
43


hsa-miR-500*
0
220
195
231
213
230
109


hsa-miR-500b
0
61
71
79
91
85
35


hsa-miR-501-3p
0
48
52
52
45
51
23


hsa-miR-501-5p
0
49
41
40
41
39
10


hsa-miR-502-3p
0
139
109
169
166
150
78


hsa-miR-502-5p
0
20
20
11
14
13
4


hsa-miR-503
0
358
309
333
280
451
53


hsa-miR-504
0
49
49
54
50
54
16


hsa-miR-505
318
238
244
291
277
287
280


hsa-miR-505*
61
33
43
56
47
39
50


hsa-miR-506
3
1390
1265
1292
1341
1382
2


hsa-miR-507
0
77
93
85
111
95
0


hsa-miR-508-3p
5
5047
4957
5494
5780
5597
15


hsa-miR-508-5p
0
90
107
74
69
123
0


hsa-miR-509-3-
0
255
201
176
142
292
1


5p









hsa-miR-509-3p
0
466
451
526
568
559
6


hsa-miR-509-5p
0
619
484
291
225
727
0


hsa-miR-510
0
296
153
136
80
346
1


hsa-miR-511
0
12
13
24
20
19
45


hsa-miR-512-3p
0
442
422
532
537
482
1


hsa-miR-512-5p
0
178
134
208
221
230
0


hsa-miR-513a-3p
0
72
46
47
50
79
0


hsa-miR-513a-5p
0
174
174
149
119
204
1


hsa-miR-513b
0
81
59
78
65
85
1


hsa-miR-513c
1
285
270
322
277
313
0


hsa-miR-514
16
12019
10822
12656
13364
13113
15


hsa-miR-514b-3p
0
53
61
72
75
62
0


hsa-miR-514b-5p
0
235
202
167
92
279
0


hsa-miR-515-3p
0
240
221
307
255
262
2


hsa-miR-515-5p
0
1542
1362
1693
1677
1619
10


hsa-miR-516a-3p
0
21
21
19
18
16
0


hsa-miR-516a-5p
0
4667
4195
4189
4168
5181
12


hsa-miR-516b
0
2726
2424
2769
2772
3014
7


hsa-miR-516b*
0
21
21
19
18
16
0


hsa-miR-517*
0
149
157
193
189
157
0


hsa-miR-517a
0
2741
2416
2981
3028
2862
20


hsa-miR-517b
0
2741
2416
2981
3028
2862
20


hsa-miR-517c
0
698
612
729
771
715
1


hsa-miR-518a-3p
0
806
768
902
828
789
4


hsa-miR-518a-5p
0
134
99
157
157
123
1


hsa-miR-518b
0
1442
1360
1575
1500
1543
14


hsa-miR-518c
0
3938
2640
3417
3452
4221
16


hsa-miR-518c*
0
62
43
52
72
69
0


hsa-miR-518d-3p
0
16
21
34
24
26
0


hsa-miR-518d-5p
0
119
128
245
271
157
2


hsa-miR-518e
0
449
466
446
421
467
2


hsa-miR-518e*
0
1853
1674
2382
2278
2063
26


hsa-miR-518f
0
930
855
925
845
980
7


hsa-miR-518f*
0
90
102
147
155
120
2


hsa-miR-519a
0
1316
1191
1350
1357
1415
3


hsa-miR-519a*
0
1853
1674
2382
2278
2063
26


hsa-miR-519b-3p
0
161
144
167
152
189
4


hsa-miR-519b-5p
0
1853
1674
2382
2278
2063
26


hsa-miR-519c-3p
0
459
433
475
443
484
8


hsa-miR-519c-5p
0
1853
1674
2382
2278
2063
26


hsa-miR-519d
0
912
750
964
967
986
7


hsa-miR-519e
0
21
17
11
20
19
0


hsa-miR-519e*
0
69
34
47
47
67
0


hsa-miR-520a-3p
0
181
148
193
179
164
1


hsa-miR-520a-5p
0
236
209
264
256
269
1


hsa-miR-520b
0
156
132
181
155
172
4


hsa-miR-520c-3p
0
156
132
181
155
172
4


hsa-miR-520c-5p
0
119
128
245
271
157
2


hsa-miR-520d-3p
0
108
134
139
131
134
0


hsa-miR-520d-5p
0
88
60
98
75
92
1


hsa-miR-520e
0
19
15
20
17
15
0


hsa-miR-520f
0
303
275
314
321
332
17


hsa-miR-520g
0
696
730
941
899
809
12


hsa-miR-520h
0
945
985
1293
1214
1076
19


hsa-miR-521
0
212
137
220
201
225
1


hsa-miR-522
0
351
323
341
381
370
1


hsa-miR-522*
0
1853
1674
2382
2278
2063
26


hsa-miR-523
0
1184
1243
1474
1434
1261
7


hsa-miR-523*
0
1853
1674
2382
2278
2063
26


hsa-miR-524-3p
0
147
112
132
104
176
2


hsa-miR-524-5p
0
152
173
179
197
188
6


hsa-miR-525-3p
0
571
547
692
584
529
1


hsa-miR-525-5p
0
368
353
400
383
384
3


hsa-miR-526a
0
119
128
245
271
157
2


hsa-miR-526b
0
602
491
683
564
599
2


hsa-miR-526b*
0
115
115
131
123
145
2


hsa-miR-527
0
134
99
157
157
123
1


hsa-miR-532-3p
0
297
291
361
366
346
238


hsa-miR-532-5p
0
1094
973
1063
1106
1140
451


hsa-miR-539
0
77
100
120
123
94
34


hsa-miR-541
0
0
1
0
0
0
1


hsa-miR-541*
1
0
0
0
1
2
0


hsa-miR-542-3p
5
2302
2186
2577
2608
2393
594


hsa-miR-542-5p
0
244
264
263
260
260
72


hsa-miR-543
0
31
47
34
41
30
6


hsa-miR-544
0
1
2
5
4
5
1


hsa-miR-544b
1
0
0
0
0
0
0


hsa-miR-545
15
1
6
7
7
14
2


hsa-miR-545*
16
3
12
11
11
7
11


hsa-miR-548a-3p
0
8
7
13
12
8
0


hsa-miR-548a-5p
0
2
0
1
2
1
1


hsa-miR-548b-3p
0
7
10
7
20
8
12


hsa-miR-548b-5p
0
0
1
2
5
8
3


hsa-miR-548c-3p
9
0
1
0
0
1
0


hsa-miR-548c-5p
6
20
23
28
29
24
9


hsa-miR-548d-3p
13
0
5
5
3
3
2


hsa-miR-548d-5p
37
23
30
45
45
32
6


hsa-miR-548e
258
30
32
18
17
41
6


hsa-miR-548f
109
4
4
7
10
5
2


hsa-miR-548g
1
0
0
0
0
0
0


hsa-miR-548h
0
0
0
2
3
1
0


hsa-miR-548i
0
7
8
12
10
12
1


hsa-miR-548j
63
15
18
14
26
10
7


hsa-miR-548k
20
16
7
11
4
18
6


hsa-miR-548l
9
5
2
4
2
4
0


hsa-miR-548m
0
0
0
0
0
0
0


hsa-miR-548n
15
4
3
4
7
4
0


hsa-miR-548o
60
12
6
11
8
12
9


hsa-miR-548p
1
0
0
1
0
1
0


hsa-miR-548q
2
4
9
6
5
4
4


hsa-miR-548s
0
0
0
2
1
1
1


hsa-miR-548t
3
0
0
1
3
4
1


hsa-miR-548u
1
0
0
1
1
0
0


hsa-miR-548v
0
1
1
2
2
2
4


hsa-miR-548w
0
1
0
0
0
0
0


hsa-miR-548x
0
0
0
0
0
0
0


hsa-miR-549
0
4
1
0
2
5
0


hsa-miR-550
0
10
16
21
12
15
11


hsa-miR-550*
3
13
11
7
6
10
7


hsa-miR-551a
2
4
0
4
1
4
2


hsa-miR-551b
0
10
6
10
17
11
34


hsa-miR-551b*
0
1
1
2
2
2
0


hsa-miR-552
2
4
2
4
2
2
0


hsa-miR-553
0
0
0
0
0
0
0


hsa-miR-554
0
0
0
0
0
0
0


hsa-miR-555
0
0
0
0
0
0
0


hsa-miR-556-3p
0
0
1
1
3
1
3


hsa-miR-556-5p
0
17
9
13
9
6
9


hsa-miR-557
0
0
0
0
0
0
0


hsa-miR-558
0
0
0
0
0
0
0


hsa-miR-559
0
1
0
2
1
0
0


hsa-miR-561
0
2
1
2
2
2
0


hsa-miR-562
0
0
0
0
0
0
0


hsa-miR-563
0
0
0
0
0
0
0


hsa-miR-564
0
0
0
1
0
0
0


hsa-miR-566
0
0
0
0
0
0
0


hsa-miR-567
0
0
0
0
0
0
0


hsa-miR-568
0
0
0
0
0
0
0


hsa-miR-569
0
0
0
0
0
0
0


hsa-miR-570
8
10
15
9
10
11
15


hsa-miR-571
0
0
0
0
0
0
0


hsa-miR-572
0
1
0
0
0
0
0


hsa-miR-573
198
0
1
0
0
0
0


hsa-miR-574-3p
0
729
814
1021
1042
847
702


hsa-miR-574-5p
0
133
123
136
124
121
169


hsa-miR-575
0
0
0
0
0
0
0


hsa-miR-576-3p
125
34
55
58
61
43
31


hsa-miR-576-5p
75
120
94
113
107
136
44


hsa-miR-577
1234
169
154
215
178
204
8


hsa-miR-578
0
0
0
0
0
0
0


hsa-miR-579
11
6
6
5
7
7
2


hsa-miR-580
55
9
7
8
10
10
0


hsa-miR-581
2
1
1
2
1
0
5


hsa-miR-582-3p
1484
121
118
137
145
135
26


hsa-miR-582-5p
592
149
183
177
151
163
35


hsa-miR-583
0
0
0
0
0
0
0


hsa-miR-584
0
108
96
109
111
104
22


hsa-miR-585
0
39
24
30
46
31
12


hsa-miR-586
0
0
0
0
0
0
0


hsa-miR-587
0
0
0
0
0
0
0


hsa-miR-588
0
0
0
0
0
0
0


hsa-miR-589
635
46
59
54
62
71
49


hsa-miR-589*
85
8
5
8
5
3
6


hsa-miR-590-3p
650
373
395
381
399
415
178


hsa-miR-590-5p
333
859
660
849
758
905
275


hsa-miR-591
0
0
0
0
0
0
0


hsa-miR-592
1
20
14
22
26
15
0


hsa-miR-593
0
0
0
0
0
0
0


hsa-miR-593*
0
0
0
0
0
0
0


hsa-miR-595
0
0
0
0
0
0
0


hsa-miR-596
0
0
0
0
0
0
0


hsa-miR-597
2
5
3
5
2
2
2


hsa-miR-598
2107
680
705
799
758
750
133


hsa-miR-599
0
0
0
0
1
0
0


hsa-miR-600
0
0
1
0
0
0
0


hsa-miR-601
0
0
0
0
0
0
0


hsa-miR-602
0
1
0
0
0
1
0


hsa-miR-603
0
1
0
0
0
0
0


hsa-miR-604
0
0
0
0
0
0
0


hsa-miR-605
0
0
0
1
4
1
0


hsa-miR-606
0
0
0
0
0
0
0


hsa-miR-607
1
0
0
0
0
0
0


hsa-miR-608
0
0
0
0
0
0
0


hsa-miR-609
0
0
0
0
1
0
0


hsa-miR-610
1
0
2
0
0
0
0


hsa-miR-611
0
0
0
0
0
0
0


hsa-miR-612
0
0
0
0
0
0
0


hsa-miR-613
0
0
0
0
0
0
0


hsa-miR-614
0
1
0
0
0
0
0


hsa-miR-615-3p
0
16
26
27
35
28
45


hsa-miR-615-5p
0
0
0
2
2
0
0


hsa-miR-616
7
3
4
3
2
5
3


hsa-miR-616*
4
23
18
20
26
30
8


hsa-miR-617
0
0
0
0
0
0
0


hsa-miR-618
0
26
25
28
30
28
31


hsa-miR-619
0
0
0
0
0
0
0


hsa-miR-620
0
0
0
0
0
0
0


hsa-miR-621
0
0
0
0
0
0
0


hsa-miR-622
0
1
1
0
0
1
0


hsa-miR-623
0
0
0
0
0
0
0


hsa-miR-624
0
3
6
2
6
4
0


hsa-miR-624*
32
14
7
9
7
9
7


hsa-miR-625
76
60
63
73
62
65
64


hsa-miR-625*
255
116
72
119
100
94
58


hsa-miR-626
0
0
0
0
0
0
0


hsa-miR-627
703
78
62
81
67
79
68


hsa-miR-628-3p
11
122
116
117
87
144
47


hsa-miR-628-5p
132
440
405
491
506
526
129


hsa-miR-629
516
36
40
43
53
46
78


hsa-miR-629*
67
10
12
10
15
17
9


hsa-miR-630
0
0
0
0
0
0
0


hsa-miR-631
0
0
0
0
0
0
0


hsa-miR-632
2
0
0
0
0
0
0


hsa-miR-633
0
0
0
0
0
0
0


hsa-miR-634
0
0
0
0
0
0
0


hsa-miR-635
0
0
0
0
0
0
0


hsa-miR-636
1
2
0
0
0
0
0


hsa-miR-637
0
0
0
0
0
0
0


hsa-miR-638
0
0
0
0
0
0
0


hsa-miR-639
1
0
0
0
1
0
0


hsa-miR-640
0
0
0
0
0
0
0


hsa-miR-641
10
10
9
7
3
13
3


hsa-miR-642
1
6
3
8
5
5
4


hsa-miR-643
12
3
2
7
10
5
6


hsa-miR-644
0
0
0
0
0
0
0


hsa-miR-645
0
0
0
0
0
0
0


hsa-miR-646
0
0
0
0
0
0
0


hsa-miR-647
0
0
0
0
0
0
0


hsa-miR-648
0
0
0
0
0
0
0


hsa-miR-649
0
0
0
0
0
0
0


hsa-miR-650
0
1
0
0
0
0
0


hsa-miR-651
372
55
60
55
54
65
42


hsa-miR-652
160
330
330
316
329
325
437


hsa-miR-653
0
43
32
37
53
39
44


hsa-miR-654-3p
0
135
128
182
161
161
102


hsa-miR-654-5p
0
5
13
12
12
12
5


hsa-miR-655
0
137
128
185
178
183
72


hsa-miR-656
0
13
19
27
16
21
18


hsa-miR-657
0
0
0
0
0
0
0


hsa-miR-658
0
0
0
0
0
0
0


hsa-miR-659
2
0
0
0
0
0
0


hsa-miR-660
0
271
259
280
290
334
171


hsa-miR-661
0
0
0
0
0
0
0


hsa-miR-662
0
0
0
0
0
0
0


hsa-miR-663
0
2
2
5
3
4
17


hsa-miR-663b
10
4
1
6
2
1
1


hsa-miR-664
483
198
174
218
213
206
152


hsa-miR-664*
98
136
127
184
177
174
200


hsa-miR-665
0
4
7
5
6
2
7


hsa-miR-668
0
1
3
2
6
5
0


hsa-miR-670
0
1
0
0
3
0
0


hsa-miR-671-3p
25
6
4
7
8
6
3


hsa-miR-671-5p
529
151
163
167
155
149
183


hsa-miR-675
0
8
7
11
2
7
5


hsa-miR-675*
0
255
247
323
283
247
115


hsa-miR-7
3569
3622
2924
2472
1896
4096
257


hsa-miR-708
0
1228
1437
1479
1584
1298
1056


hsa-miR-708*
0
68
48
79
76
72
35


hsa-miR-7-1*
295
64
57
58
57
35
22


hsa-miR-711
0
0
0
0
0
0
0


hsa-miR-718
0
0
0
0
0
0
0


hsa-miR-7-2*
0
12
5
9
6
7
4


hsa-miR-720
154
188
204
273
188
188
30


hsa-miR-744
862
707
698
750
701
733
628


hsa-miR-744*
20
27
21
32
24
15
11


hsa-miR-758
0
5
7
7
8
6
2


hsa-miR-759
0
0
0
0
0
0
0


hsa-miR-760
1
5
9
4
6
6
10


hsa-miR-761
0
0
0
0
0
0
0


hsa-miR-762
0
0
0
0
0
0
0


hsa-miR-764
0
0
0
0
0
0
0


hsa-miR-765
0
3
1
3
3
6
1


hsa-miR-766
229
87
73
93
89
110
29


hsa-miR-767-3p
0
2
0
0
1
0
0


hsa-miR-767-5p
0
3
10
7
11
5
0


hsa-miR-769-3p
91
5
6
10
8
6
3


hsa-miR-769-5p
4023
531
514
547
553
547
464


hsa-miR-770-5p
0
1
4
1
3
7
0


hsa-miR-802
0
46
50
53
51
52
0


hsa-miR-873
0
19
19
17
11
26
2


hsa-miR-874
10
234
230
307
286
254
193


hsa-miR-875-3p
0
0
0
0
0
0
0


hsa-miR-875-5p
0
5
1
0
2
0
1


hsa-miR-876-3p
0
1
0
1
0
4
0


hsa-miR-876-5p
0
3
1
6
2
1
1


hsa-miR-877
846
133
104
75
55
151
96


hsa-miR-877*
2
1
0
0
1
0
0


hsa-miR-885-3p
0
14
12
8
8
6
1


hsa-miR-885-5p
0
89
94
66
93
84
2


hsa-miR-886-3p
69
5
12
12
8
6
8


hsa-miR-886-5p
15314
661
618
705
721
710
1271


hsa-miR-887
0
108
132
128
127
131
116


hsa-miR-888
0
55
49
58
75
84
0


hsa-miR-888*
0
0
1
2
0
1
0


hsa-miR-889
0
211
180
273
254
234
57


hsa-miR-890
0
4
1
5
6
11
0


hsa-miR-891a
1
358
311
363
390
384
4


hsa-miR-891b
0
14
10
11
8
4
0


hsa-miR-892a
0
7
4
10
7
6
0


hsa-miR-892b
0
5
5
5
2
6
0


hsa-miR-9
13310
10506
9814
11411
11015
11489
233


hsa-miR-9*
1880
2040
2327
2671
2670
2212
75


hsa-miR-920
0
0
0
0
0
0
0


hsa-miR-921
0
0
0
0
0
0
0


hsa-miR-922
0
0
0
0
0
0
0


hsa-miR-924
0
0
0
0
0
0
0


hsa-miR-92a
27036
4490
4235
4907
4803
4907
3961


hsa-miR-92a-1*
328
61
43
71
61
47
42


hsa-miR-92a-2*
0
0
2
1
1
0
0


hsa-miR-92b
65
508
522
615
602
591
314


hsa-miR-92b*
4
25
17
15
13
25
9


hsa-miR-93
29354
5197
4866
5765
5546
5717
4574


hsa-miR-93*
25
39
37
39
37
38
71


hsa-miR-933
0
0
0
0
0
1
0


hsa-miR-934
0
101
149
198
206
145
49


hsa-miR-935
1
74
104
125
114
94
9


hsa-miR-936
0
0
0
0
0
0
0


hsa-miR-937
1
0
2
0
0
0
0


hsa-miR-938
0
0
0
0
0
0
0


hsa-miR-939
8
1
0
0
0
0
0


hsa-miR-940
7
4
7
8
5
10
4


hsa-miR-941
244
145
116
136
116
173
43


hsa-miR-942
75
8
12
12
11
8
3


hsa-miR-943
1
0
0
0
0
0
2


hsa-miR-944
1109
143
143
144
158
164
26


hsa-miR-95
57
3409
3489
3620
3782
3901
720


hsa-miR-96
783
146
122
146
138
173
681


hsa-miR-96*
0
0
0
0
0
0
1


hsa-miR-98
1602
2924
2956
3366
3559
3153
2291


hsa-miR-99a
692
23127
23513
27764
27299
25367
12696


hsa-miR-99a*
42
116
97
118
115
112
68


hsa-miR-99b
9
16328
14746
17477
16934
17389
6543


hsa-miR-99b*
0
52
58
73
42
55
51



















TABLE 11







miRNA
Read count-all



















hsa-let-7a
3263253



hsa-let-7a*
2627



hsa-let-7a-2*
130



hsa-let-7b
629946



hsa-let-7b*
2206



hsa-let-7c
586231



hsa-let-7c*
41



hsa-let-7d
80917



hsa-let-7d*
13259



hsa-let-7e
115027



hsa-let-7e*
774



hsa-let-7f
3480770



hsa-let-7f-1*
344



hsa-let-7f-2*
144



hsa-let-7g
729933



hsa-let-7g*
2239



hsa-let-7i
123591



hsa-let-7i*
3761



hsa-miR-1
4827651



hsa-miR-100
371538



hsa-miR-100*
359



hsa-miR-101
1018648



hsa-miR-101*
4418



hsa-miR-103
608126



hsa-miR-103-2*
301



hsa-miR-103-as
0



hsa-miR-105
154



hsa-miR-105*
6



hsa-miR-106a
17036



hsa-miR-106a*
87



hsa-miR-106b
76043



hsa-miR-106b*
3665



hsa-miR-107
5450



hsa-miR-10a
411798



hsa-miR-10a*
1392



hsa-miR-10b
671361



hsa-miR-10b*
4311



hsa-miR-1178
0



hsa-miR-1179
977



hsa-miR-1180
351



hsa-miR-1181
1



hsa-miR-1182
0



hsa-miR-1183
0



hsa-miR-1184
0



hsa-miR-1185
503



hsa-miR-1193
3



hsa-miR-1197
20



hsa-miR-1200
0



hsa-miR-1201
1283



hsa-miR-1202
0



hsa-miR-1203
0



hsa-miR-1204
0



hsa-miR-1205
0



hsa-miR-1206
0



hsa-miR-1207-3p
0



hsa-miR-1207-5p
0



hsa-miR-1208
0



hsa-miR-122
71506



hsa-miR-122*
881



hsa-miR-1224-3p
8



hsa-miR-1224-5p
212



hsa-miR-1225-3p
2



hsa-miR-1225-5p
12



hsa-miR-1226
35



hsa-miR-1226*
11



hsa-miR-1227
0



hsa-miR-1228
10



hsa-miR-1228*
9



hsa-miR-1229
15



hsa-miR-1231
9



hsa-miR-1233
5



hsa-miR-1234
7



hsa-miR-1236
0



hsa-miR-1237
0



hsa-miR-1238
1



hsa-miR-124
14855



hsa-miR-124*
55



hsa-miR-1243
28



hsa-miR-1244
43



hsa-miR-1245
23



hsa-miR-1246
163980



hsa-miR-1247
1190



hsa-miR-1248
266



hsa-miR-1249
348



hsa-miR-1250
22



hsa-miR-1251
956



hsa-miR-1252
16



hsa-miR-1253
0



hsa-miR-1254
114



hsa-miR-1255a
204



hsa-miR-1255b
59



hsa-miR-1256
17



hsa-miR-1257
29



hsa-miR-1258
38



hsa-miR-1259
807



hsa-miR-125a-3p
820



hsa-miR-125a-5p
197714



hsa-miR-125b
407709



hsa-miR-125b-1*
966



hsa-miR-125b-2*
18552



hsa-miR-126
2107272



hsa-miR-126*
279706



hsa-miR-1260
16



hsa-miR-1260b
216



hsa-miR-1261
0



hsa-miR-1262
184



hsa-miR-1263
2



hsa-miR-1264
13



hsa-miR-1265
5



hsa-miR-1266
61



hsa-miR-1267
2



hsa-miR-1268
788



hsa-miR-1269
157



hsa-miR-1270
1259



hsa-miR-1271
576



hsa-miR-1272
3



hsa-miR-1273
0



hsa-miR-1273c
91



hsa-miR-1273d
9



hsa-miR-127-3p
27435



hsa-miR-1274a
15



hsa-miR-1274b
617



hsa-miR-1275
746



hsa-miR-127-5p
4243



hsa-miR-1276
36



hsa-miR-1277
467



hsa-miR-1278
34



hsa-miR-1279
0



hsa-miR-128
28463



hsa-miR-1280
75



hsa-miR-1281
0



hsa-miR-1282
0



hsa-miR-1283
3052



hsa-miR-1284
12



hsa-miR-1285
568



hsa-miR-1286
6



hsa-miR-1287
415



hsa-miR-1288
3



hsa-miR-1289
1



hsa-miR-129*
332



hsa-miR-1290
6



hsa-miR-1291
364



hsa-miR-1292
518



hsa-miR-1293
2



hsa-miR-129-3p
1131



hsa-miR-1294
41



hsa-miR-1295
27



hsa-miR-129-5p
4027



hsa-miR-1296
785



hsa-miR-1297
0



hsa-miR-1298
246



hsa-miR-1299
935



hsa-miR-1301
608



hsa-miR-1302
1



hsa-miR-1303
133



hsa-miR-1304
50



hsa-miR-1305
55



hsa-miR-1306
33



hsa-miR-1307
6293



hsa-miR-1308
7471



hsa-miR-130a
30830



hsa-miR-130a*
43



hsa-miR-130b
8344



hsa-miR-130b*
755



hsa-miR-132
10033



hsa-miR-132*
1469



hsa-miR-1321
2



hsa-miR-1322
12



hsa-miR-1323
40121



hsa-miR-1324
0



hsa-miR-133a
199457



hsa-miR-133b
6363



hsa-miR-134
3019



hsa-miR-135a
108493



hsa-miR-135a*
121



hsa-miR-135b
33044



hsa-miR-135b*
155



hsa-miR-136
21356



hsa-miR-136*
5945



hsa-miR-137
6124



hsa-miR-138
16603



hsa-miR-138-1*
503



hsa-miR-138-2*
50



hsa-miR-139-3p
1864



hsa-miR-139-5p
19160



hsa-miR-140-3p
632569



hsa-miR-140-5p
99250



hsa-miR-141
87549



hsa-miR-141*
1759



hsa-miR-142-3p
863170



hsa-miR-142-5p
40824



hsa-miR-143
27191001



hsa-miR-143*
131103



hsa-miR-144
13924



hsa-miR-144*
141389



hsa-miR-145
810044



hsa-miR-145*
32400



hsa-miR-1468
68



hsa-miR-1469
0



hsa-miR-146a
159091



hsa-miR-146a*
24



hsa-miR-146b-3p
431



hsa-miR-146b-5p
945206



hsa-miR-147
0



hsa-miR-1470
0



hsa-miR-1471
0



hsa-miR-147b
90



hsa-miR-148a
1306692



hsa-miR-148a*
6159



hsa-miR-148b
188242



hsa-miR-148b*
7002



hsa-miR-149
2802



hsa-miR-149*
49



hsa-miR-150
26608



hsa-miR-150*
259



hsa-miR-151-3p
142414



hsa-miR-151-5p
135256



hsa-miR-152
179559



hsa-miR-153
3912



hsa-miR-1537
22



hsa-miR-1538
2



hsa-miR-1539
0



hsa-miR-154
758



hsa-miR-154*
270



hsa-miR-155
162738



hsa-miR-155*
497



hsa-miR-15a
45924



hsa-miR-15a*
47



hsa-miR-15b
18241



hsa-miR-15b*
1885



hsa-miR-16
434371



hsa-miR-16-1*
327



hsa-miR-16-2*
1409



hsa-miR-17
176460



hsa-miR-17*
12954



hsa-miR-181a
345345



hsa-miR-181a*
4960



hsa-miR-181a-2*
4066



hsa-miR-181b
49597



hsa-miR-181c
6636



hsa-miR-181c*
1044



hsa-miR-181d
4815



hsa-miR-182
97847



hsa-miR-182*
5



hsa-miR-1825
0



hsa-miR-1826
3477



hsa-miR-1827
1



hsa-miR-183
22483



hsa-miR-183*
92



hsa-miR-184
4415



hsa-miR-185
13309



hsa-miR-185*
199



hsa-miR-186
148992



hsa-miR-186*
433



hsa-miR-187
790



hsa-miR-187*
57



hsa-miR-188-3p
43



hsa-miR-188-5p
633



hsa-miR-18a
14206



hsa-miR-18a*
302



hsa-miR-18b
147



hsa-miR-18b*
17



hsa-miR-190
13151



hsa-miR-1908
14



hsa-miR-1909
1



hsa-miR-1909*
0



hsa-miR-190b
526



hsa-miR-191
347191



hsa-miR-191*
185



hsa-miR-1910
2



hsa-miR-1911
149



hsa-miR-1911*
1



hsa-miR-1912
21



hsa-miR-1913
2



hsa-miR-1914
1



hsa-miR-1914*
1



hsa-miR-1915
2



hsa-miR-1915*
0



hsa-miR-192
915301



hsa-miR-192*
1029



hsa-miR-193a-3p
3073



hsa-miR-193a-5p
4589



hsa-miR-193b
29643



hsa-miR-193b*
1332



hsa-miR-194
338472



hsa-miR-194*
572



hsa-miR-195
76407



hsa-miR-195*
464



hsa-miR-196a
8449



hsa-miR-196a*
33



hsa-miR-196b
20661



hsa-miR-196b*
27



hsa-miR-197
15658



hsa-miR-1972
0



hsa-miR-1973
128



hsa-miR-1975
44554



hsa-miR-1976
89



hsa-miR-1979
42616



hsa-miR-198
2



hsa-miR-199a-3p
267443



hsa-miR-199a-5p
58426



hsa-miR-199b-3p
267443



hsa-miR-199b-5p
15208



hsa-miR-19a
28904



hsa-miR-19a*
27



hsa-miR-19b
117578



hsa-miR-19b-1*
545



hsa-miR-19b-2*
2



hsa-miR-200a
30201



hsa-miR-200a*
735



hsa-miR-200b
52688



hsa-miR-200b*
4943



hsa-miR-200c
427036



hsa-miR-200c*
127



hsa-miR-202
6243



hsa-miR-202*
91648



hsa-miR-203
73346



hsa-miR-204
8758



hsa-miR-205
121249



hsa-miR-205*
21



hsa-miR-2052
0



hsa-miR-2053
0



hsa-miR-2054
0



hsa-miR-206
239800



hsa-miR-208a
457



hsa-miR-208b
3831



hsa-miR-20a
261873



hsa-miR-20a*
4565



hsa-miR-20b
14523



hsa-miR-20b*
181



hsa-miR-21
6223972



hsa-miR-21*
4482



hsa-miR-210
14772



hsa-miR-211
31



hsa-miR-2110
1305



hsa-miR-2113
0



hsa-miR-2114
93



hsa-miR-2114*
11



hsa-miR-2115
46



hsa-miR-2115*
85



hsa-miR-2116
32



hsa-miR-2116*
33



hsa-miR-2117
0



hsa-miR-212
317



hsa-miR-214
13273



hsa-miR-214*
2118



hsa-miR-215
848606



hsa-miR-216a
260



hsa-miR-216b
705



hsa-miR-217
754



hsa-miR-218
22992



hsa-miR-218-1*
624



hsa-miR-218-2*
26



hsa-miR-219-1-3p
108



hsa-miR-219-2-3p
26649



hsa-miR-219-5p
500



hsa-miR-22
159941



hsa-miR-22*
12506



hsa-miR-220a
0



hsa-miR-220b
0



hsa-miR-220c
0



hsa-miR-221
44792



hsa-miR-221*
17403



hsa-miR-222
94321



hsa-miR-222*
430



hsa-miR-223
37671



hsa-miR-223*
1142



hsa-miR-224
8474



hsa-miR-224*
438



hsa-miR-2276
105



hsa-miR-2277
30



hsa-miR-2278
12



hsa-miR-2355
80



hsa-miR-23a
243206



hsa-miR-23a*
23



hsa-miR-23b
210543



hsa-miR-23b*
231



hsa-miR-24
1502194



hsa-miR-24-1*
3733



hsa-miR-24-2*
6950



hsa-miR-25
33806



hsa-miR-25*
649



hsa-miR-26a
1986940



hsa-miR-26a-1*
62



hsa-miR-26a-2*
834



hsa-miR-26b
482254



hsa-miR-26b*
679



hsa-miR-27a
267030



hsa-miR-27a*
745



hsa-miR-27b
673420



hsa-miR-27b*
8575



hsa-miR-28-3p
52126



hsa-miR-28-5p
60747



hsa-miR-2861
0



hsa-miR-2909
0



hsa-miR-296-3p
116



hsa-miR-296-5p
465



hsa-miR-297
0



hsa-miR-298
0



hsa-miR-299-3p
427



hsa-miR-299-5p
860



hsa-miR-29a
758430



hsa-miR-29a*
4199



hsa-miR-29b
224003



hsa-miR-29b-1*
332



hsa-miR-29b-2*
1741



hsa-miR-29c
50309



hsa-miR-29c*
5043



hsa-miR-300
0



hsa-miR-301a
1455



hsa-miR-301b
289



hsa-miR-302a
34



hsa-miR-302a*
132



hsa-miR-302b
81



hsa-miR-302b*
2



hsa-miR-302c
11



hsa-miR-302c*
0



hsa-miR-302d
23



hsa-miR-302d*
0



hsa-miR-302e
0



hsa-miR-302f
0



hsa-miR-3065-3p
1456



hsa-miR-3065-5p
1144



hsa-miR-3074
18



hsa-miR-30a
2082964



hsa-miR-30a*
90594



hsa-miR-30b
253420



hsa-miR-30b*
583



hsa-miR-30c
363593



hsa-miR-30c-1*
432



hsa-miR-30c-2*
5633



hsa-miR-30d
1167397



hsa-miR-30d*
1597



hsa-miR-30e
888607



hsa-miR-30e*
96423



hsa-miR-31
11912



hsa-miR-31*
370



hsa-miR-3115
19



hsa-miR-3116
5



hsa-miR-3117
116



hsa-miR-3118
1



hsa-miR-3119
0



hsa-miR-3120
8



hsa-miR-3121
21



hsa-miR-3122
4



hsa-miR-3123
0



hsa-miR-3124
46



hsa-miR-3125
32



hsa-miR-3126-3p
5



hsa-miR-3126-5p
7



hsa-miR-3127
53



hsa-miR-3128
30



hsa-miR-3129
81



hsa-miR-3130-3p
1



hsa-miR-3130-5p
31



hsa-miR-3131
9



hsa-miR-3132
7



hsa-miR-3133
18



hsa-miR-3134
12



hsa-miR-3135
4



hsa-miR-3136
15



hsa-miR-3137
0



hsa-miR-3138
22



hsa-miR-3139
9



hsa-miR-3140
19



hsa-miR-3141
9



hsa-miR-3142
0



hsa-miR-3143
9



hsa-miR-3144-3p
6



hsa-miR-3144-5p
11



hsa-miR-3145
13



hsa-miR-3146
34



hsa-miR-3147
0



hsa-miR-3148
1



hsa-miR-3149
27



hsa-miR-3150
1



hsa-miR-3151
2



hsa-miR-3152
1



hsa-miR-3153
0



hsa-miR-3154
129



hsa-miR-3155
2



hsa-miR-3156
1



hsa-miR-3157
22



hsa-miR-3158
221



hsa-miR-3159
6



hsa-miR-3160
4



hsa-miR-3161
11



hsa-miR-3162
1



hsa-miR-3163
21



hsa-miR-3164
60



hsa-miR-3165
0



hsa-miR-3166
2



hsa-miR-3167
6



hsa-miR-3168
4



hsa-miR-3169
0



hsa-miR-3170
5



hsa-miR-3171
2



hsa-miR-3172
729



hsa-miR-3173
9



hsa-miR-3174
21



hsa-miR-3175
10



hsa-miR-3176
59



hsa-miR-3177
63



hsa-miR-3178
0



hsa-miR-3179
23



hsa-miR-3180-3p
14



hsa-miR-3180-5p
10



hsa-miR-3181
5



hsa-miR-3182
11



hsa-miR-3183
1



hsa-miR-3184
2



hsa-miR-3185
0



hsa-miR-3186-3p
0



hsa-miR-3186-5p
1



hsa-miR-3187
25



hsa-miR-3188
118



hsa-miR-3189
2



hsa-miR-3190-3p
22



hsa-miR-3190-5p
22



hsa-miR-3191
5



hsa-miR-3192
11



hsa-miR-3193
7



hsa-miR-3194
52



hsa-miR-3195
6



hsa-miR-3196
10



hsa-miR-3197
0



hsa-miR-3198
1



hsa-miR-3199
62



hsa-miR-32
25140



hsa-miR-32*
301



hsa-miR-3200
515



hsa-miR-3201
0



hsa-miR-3202
28



hsa-miR-320a
111802



hsa-miR-320b
8719



hsa-miR-320c
5424



hsa-miR-320d
5980



hsa-miR-320e
241



hsa-miR-323-3p
662



hsa-miR-323-5p
8



hsa-miR-323b-3p
216



hsa-miR-323b-5p
2



hsa-miR-324-3p
1220



hsa-miR-324-5p
2258



hsa-miR-325
0



hsa-miR-326
199



hsa-miR-328
2414



hsa-miR-329
1531



hsa-miR-330-3p
4705



hsa-miR-330-5p
751



hsa-miR-331-3p
1768



hsa-miR-331-5p
637



hsa-miR-335
38506



hsa-miR-335*
7818



hsa-miR-337-3p
3906



hsa-miR-337-5p
4909



hsa-miR-338-3p
8532



hsa-miR-338-5p
5359



hsa-miR-339-3p
10563



hsa-miR-339-5p
2593



hsa-miR-33a
13217



hsa-miR-33a*
371



hsa-miR-33b
1119



hsa-miR-33b*
78



hsa-miR-340
28380



hsa-miR-340*
2305



hsa-miR-342-3p
24621



hsa-miR-342-5p
2668



hsa-miR-345
3394



hsa-miR-346
108



hsa-miR-34a
13143



hsa-miR-34a*
92



hsa-miR-34b
1191



hsa-miR-34b*
1105



hsa-miR-34c-3p
1826



hsa-miR-34c-5p
38125



hsa-miR-361-3p
3940



hsa-miR-361-5p
21423



hsa-miR-362-3p
739



hsa-miR-362-5p
2360



hsa-miR-363
16685



hsa-miR-363*
24



hsa-miR-365
20092



hsa-miR-365*
205



hsa-miR-367
15



hsa-miR-367*
0



hsa-miR-369-3p
6883



hsa-miR-369-5p
1237



hsa-miR-370
726



hsa-miR-371-3p
22



hsa-miR-371-5p
132



hsa-miR-372
421



hsa-miR-373
280



hsa-miR-373*
16



hsa-miR-374a
51385



hsa-miR-374a*
114379



hsa-miR-374b
39066



hsa-miR-374b*
1323



hsa-miR-375
22890



hsa-miR-376a
1004



hsa-miR-376a*
1912



hsa-miR-376b
1253



hsa-miR-376c
27443



hsa-miR-377
4408



hsa-miR-377*
75



hsa-miR-378
4360229



hsa-miR-378*
2959



hsa-miR-378b
33



hsa-miR-378c
43898



hsa-miR-379
46853



hsa-miR-379*
210



hsa-miR-380
122



hsa-miR-380*
491



hsa-miR-381
18003



hsa-miR-382
2040



hsa-miR-383
829



hsa-miR-384
0



hsa-miR-409-3p
1561



hsa-miR-409-5p
477



hsa-miR-410
1428



hsa-miR-411
12443



hsa-miR-411*
534



hsa-miR-412
1



hsa-miR-421
2693



hsa-miR-422a
11



hsa-miR-423-3p
37223



hsa-miR-423-5p
83406



hsa-miR-424
209609



hsa-miR-424*
2020



hsa-miR-425
77674



hsa-miR-425*
3099



hsa-miR-4251
0



hsa-miR-4252
0



hsa-miR-4253
0



hsa-miR-4254
4



hsa-miR-4255
0



hsa-miR-4256
0



hsa-miR-4257
0



hsa-miR-4258
0



hsa-miR-4259
0



hsa-miR-4260
0



hsa-miR-4261
0



hsa-miR-4262
0



hsa-miR-4263
0



hsa-miR-4264
0



hsa-miR-4265
0



hsa-miR-4266
0



hsa-miR-4267
0



hsa-miR-4268
0



hsa-miR-4269
0



hsa-miR-4270
0



hsa-miR-4271
0



hsa-miR-4272
0



hsa-miR-4273
0



hsa-miR-4274
0



hsa-miR-4275
0



hsa-miR-4276
0



hsa-miR-4277
0



hsa-miR-4278
0



hsa-miR-4279
0



hsa-miR-4280
0



hsa-miR-4281
0



hsa-miR-4282
0



hsa-miR-4283
0



hsa-miR-4284
1392



hsa-miR-4285
0



hsa-miR-4286
1480



hsa-miR-4287
0



hsa-miR-4288
0



hsa-miR-4289
0



hsa-miR-429
21310



hsa-miR-4290
0



hsa-miR-4291
0



hsa-miR-4292
0



hsa-miR-4293
0



hsa-miR-4294
0



hsa-miR-4295
0



hsa-miR-4296
1



hsa-miR-4297
0



hsa-miR-4298
0



hsa-miR-4299
0



hsa-miR-4300
0



hsa-miR-4301
0



hsa-miR-4302
0



hsa-miR-4303
0



hsa-miR-4304
0



hsa-miR-4305
0



hsa-miR-4306
6



hsa-miR-4307
0



hsa-miR-4308
0



hsa-miR-4309
0



hsa-miR-431
128



hsa-miR-431*
145



hsa-miR-4310
0



hsa-miR-4311
0



hsa-miR-4312
0



hsa-miR-4313
0



hsa-miR-4314
0



hsa-miR-4315
0



hsa-miR-4316
0



hsa-miR-4317
0



hsa-miR-4318
0



hsa-miR-4319
0



hsa-miR-432
1080



hsa-miR-432*
10



hsa-miR-4320
0



hsa-miR-4321
0



hsa-miR-4322
0



hsa-miR-4323
1



hsa-miR-4324
0



hsa-miR-4325
0



hsa-miR-4326
73



hsa-miR-4327
0



hsa-miR-4328
27



hsa-miR-4329
0



hsa-miR-433
529



hsa-miR-4330
0



hsa-miR-448
8



hsa-miR-449a
8663



hsa-miR-449b
1369



hsa-miR-449b*
29



hsa-miR-449c
2070



hsa-miR-449c*
0



hsa-miR-450a
10118



hsa-miR-450b-3p
32



hsa-miR-450b-5p
4838



hsa-miR-451
737681



hsa-miR-452
104661



hsa-miR-452*
2187



hsa-miR-454
16148



hsa-miR-454*
111



hsa-miR-455-3p
14394



hsa-miR-455-5p
3633



hsa-miR-466
3



hsa-miR-483-3p
395



hsa-miR-483-5p
3635



hsa-miR-484
2717



hsa-miR-485-3p
545



hsa-miR-485-5p
1942



hsa-miR-486-3p
296



hsa-miR-486-5p
12961



hsa-miR-487a
707



hsa-miR-487b
3131



hsa-miR-488
328



hsa-miR-488*
26



hsa-miR-489
644



hsa-miR-490-3p
1085



hsa-miR-490-5p
906



hsa-miR-491-3p
97



hsa-miR-491-5p
761



hsa-miR-492
6



hsa-miR-493
1152



hsa-miR-493*
2341



hsa-miR-494
2613



hsa-miR-495
2968



hsa-miR-496
93



hsa-miR-497
9453



hsa-miR-497*
249



hsa-miR-498
1074



hsa-miR-499-3p
7353



hsa-miR-499-5p
16967



hsa-miR-500
1116



hsa-miR-500*
3156



hsa-miR-500b
907



hsa-miR-501-3p
736



hsa-miR-501-5p
546



hsa-miR-502-3p
1821



hsa-miR-502-5p
188



hsa-miR-503
5056



hsa-miR-504
637



hsa-miR-505
3599



hsa-miR-505*
598



hsa-miR-506
16376



hsa-miR-507
840



hsa-miR-508-3p
76162



hsa-miR-508-5p
1212



hsa-miR-509-3-5p
3903



hsa-miR-509-3p
7241



hsa-miR-509-5p
9245



hsa-miR-510
1916



hsa-miR-511
277



hsa-miR-512-3p
5968



hsa-miR-512-5p
1757



hsa-miR-513a-3p
544



hsa-miR-513a-5p
2002



hsa-miR-513b
1058



hsa-miR-513c
3568



hsa-miR-514
156995



hsa-miR-514b-3p
711



hsa-miR-514b-5p
2112



hsa-miR-515-3p
2866



hsa-miR-515-5p
18861



hsa-miR-516a-3p
263



hsa-miR-516a-5p
56271



hsa-miR-516b
32784



hsa-miR-516b*
263



hsa-miR-517*
1742



hsa-miR-517a
34727



hsa-miR-517b
34727



hsa-miR-517c
8109



hsa-miR-518a-3p
8007



hsa-miR-518a-5p
1202



hsa-miR-518b
16097



hsa-miR-518c
33419



hsa-miR-518c*
610



hsa-miR-518d-3p
262



hsa-miR-518d-5p
1426



hsa-miR-518e
5542



hsa-miR-518e*
17339



hsa-miR-518f
10142



hsa-miR-518f*
1041



hsa-miR-519a
16658



hsa-miR-519a*
17339



hsa-miR-519b-3p
2282



hsa-miR-519b-5p
17339



hsa-miR-519c-3p
5238



hsa-miR-519c-5p
17339



hsa-miR-519d
9568



hsa-miR-519e
190



hsa-miR-519e*
494



hsa-miR-520a-3p
2395



hsa-miR-520a-5p
2644



hsa-miR-520b
1691



hsa-miR-520c-3p
1691



hsa-miR-520c-5p
1426



hsa-miR-520d-3p
1408



hsa-miR-520d-5p
841



hsa-miR-520e
205



hsa-miR-520f
3634



hsa-miR-520g
7961



hsa-miR-520h
10836



hsa-miR-521
2359



hsa-miR-522
4224



hsa-miR-522*
17339



hsa-miR-523
16402



hsa-miR-523*
17339



hsa-miR-524-3p
1566



hsa-miR-524-5p
1943



hsa-miR-525-3p
7310



hsa-miR-525-5p
4442



hsa-miR-526a
1426



hsa-miR-526b
6191



hsa-miR-526b*
1651



hsa-miR-527
1202



hsa-miR-532-3p
3688



hsa-miR-532-5p
14360



hsa-miR-539
1024



hsa-miR-541
13



hsa-miR-541*
9



hsa-miR-542-3p
30135



hsa-miR-542-5p
2681



hsa-miR-543
511



hsa-miR-544
30



hsa-miR-544b
2



hsa-miR-545
84



hsa-miR-545*
146



hsa-miR-548a-3p
133



hsa-miR-548a-5p
15



hsa-miR-548b-3p
102



hsa-miR-548b-5p
40



hsa-miR-548c-3p
18



hsa-miR-548c-5p
290



hsa-miR-548d-3p
46



hsa-miR-548d-5p
467



hsa-miR-548e
754



hsa-miR-548f
220



hsa-miR-548g
1



hsa-miR-548h
18



hsa-miR-548i
106



hsa-miR-548j
242



hsa-miR-548k
210



hsa-miR-548l
56



hsa-miR-548m
0



hsa-miR-548n
55



hsa-miR-548o
231



hsa-miR-548p
5



hsa-miR-548q
79



hsa-miR-548s
11



hsa-miR-548t
19



hsa-miR-548u
4



hsa-miR-548v
46



hsa-miR-548w
3



hsa-miR-548x
0



hsa-miR-549
26



hsa-miR-550
183



hsa-miR-550*
125



hsa-miR-551a
28



hsa-miR-551b
129



hsa-miR-551b*
31



hsa-miR-552
61



hsa-miR-553
0



hsa-miR-554
0



hsa-miR-555
0



hsa-miR-556-3p
24



hsa-miR-556-5p
141



hsa-miR-557
0



hsa-miR-558
0



hsa-miR-559
6



hsa-miR-561
24



hsa-miR-562
0



hsa-miR-563
0



hsa-miR-564
1



hsa-miR-566
0



hsa-miR-567
0



hsa-miR-568
0



hsa-miR-569
0



hsa-miR-570
146



hsa-miR-571
0



hsa-miR-572
1



hsa-miR-573
199



hsa-miR-574-3p
10204



hsa-miR-574-5p
1628



hsa-miR-575
0



hsa-miR-576-3p
683



hsa-miR-576-5p
1574



hsa-miR-577
3344



hsa-miR-578
0



hsa-miR-579
63



hsa-miR-580
150



hsa-miR-581
17



hsa-miR-582-3p
3511



hsa-miR-582-5p
2439



hsa-miR-583
0



hsa-miR-584
1494



hsa-miR-585
436



hsa-miR-586
0



hsa-miR-587
0



hsa-miR-588
0



hsa-miR-589
1443



hsa-miR-589*
155



hsa-miR-590-3p
5086



hsa-miR-590-5p
11304



hsa-miR-591
0



hsa-miR-592
269



hsa-miR-593
0



hsa-miR-593*
0



hsa-miR-595
0



hsa-miR-596
0



hsa-miR-597
33



hsa-miR-598
10797



hsa-miR-599
2



hsa-miR-600
9



hsa-miR-601
0



hsa-miR-602
3



hsa-miR-603
5



hsa-miR-604
0



hsa-miR-605
28



hsa-miR-606
0



hsa-miR-607
1



hsa-miR-608
0



hsa-miR-609
2



hsa-miR-610
4



hsa-miR-611
0



hsa-miR-612
0



hsa-miR-613
0



hsa-miR-614
2



hsa-miR-615-3p
341



hsa-miR-615-5p
4



hsa-miR-616
57



hsa-miR-616*
196



hsa-miR-617
0



hsa-miR-618
356



hsa-miR-619
0



hsa-miR-620
0



hsa-miR-621
0



hsa-miR-622
10



hsa-miR-623
0



hsa-miR-624
47



hsa-miR-624*
142



hsa-miR-625
791



hsa-miR-625*
1449



hsa-miR-626
0



hsa-miR-627
1704



hsa-miR-628-3p
1869



hsa-miR-628-5p
6569



hsa-miR-629
1282



hsa-miR-629*
187



hsa-miR-630
0



hsa-miR-631
0



hsa-miR-632
2



hsa-miR-633
0



hsa-miR-634
0



hsa-miR-635
0



hsa-miR-636
6



hsa-miR-637
0



hsa-miR-638
0



hsa-miR-639
3



hsa-miR-640
0



hsa-miR-641
122



hsa-miR-642
81



hsa-miR-643
69



hsa-miR-644
0



hsa-miR-645
0



hsa-miR-646
0



hsa-miR-647
0



hsa-miR-648
0



hsa-miR-649
0



hsa-miR-650
3



hsa-miR-651
1075



hsa-miR-652
4967



hsa-miR-653
479



hsa-miR-654-3p
2161



hsa-miR-654-5p
151



hsa-miR-655
1774



hsa-miR-656
274



hsa-miR-657
0



hsa-miR-658
1



hsa-miR-659
2



hsa-miR-660
3414



hsa-miR-661
0



hsa-miR-662
0



hsa-miR-663
60



hsa-miR-663b
44



hsa-miR-664
3101



hsa-miR-664*
2320



hsa-miR-665
65



hsa-miR-668
53



hsa-miR-670
5



hsa-miR-671-3p
92



hsa-miR-671-5p
2990



hsa-miR-675
71



hsa-miR-675*
2472



hsa-miR-7
66615



hsa-miR-708
16305



hsa-miR-708*
1017



hsa-miR-7-1*
976



hsa-miR-711
0



hsa-miR-718
0



hsa-miR-7-2*
86



hsa-miR-720
2724



hsa-miR-744
10452



hsa-miR-744*
248



hsa-miR-758
89



hsa-miR-759
0



hsa-miR-760
95



hsa-miR-761
0



hsa-miR-762
0



hsa-miR-764
0



hsa-miR-765
46



hsa-miR-766
1204



hsa-miR-767-3p
5



hsa-miR-767-5p
72



hsa-miR-769-3p
163



hsa-miR-769-5p
11515



hsa-miR-770-5p
26



hsa-miR-802
486



hsa-miR-873
305



hsa-miR-874
2982



hsa-miR-875-3p
0



hsa-miR-875-5p
15



hsa-miR-876-3p
25



hsa-miR-876-5p
19



hsa-miR-877
2724



hsa-miR-877*
9



hsa-miR-885-3p
173



hsa-miR-885-5p
906



hsa-miR-886-3p
168



hsa-miR-886-5p
27435



hsa-miR-887
1793



hsa-miR-888
666



hsa-miR-888*
7



hsa-miR-889
3236



hsa-miR-890
111



hsa-miR-891a
4506



hsa-miR-891b
94



hsa-miR-892a
87



hsa-miR-892b
49



hsa-miR-9
165442



hsa-miR-9*
29721



hsa-miR-920
0



hsa-miR-921
1



hsa-miR-922
0



hsa-miR-924
0



hsa-miR-92a
85398



hsa-miR-92a-1*
1103



hsa-miR-92a-2*
6



hsa-miR-92b
6766



hsa-miR-92b*
391



hsa-miR-93
95298



hsa-miR-93*
475



hsa-miR-933
2



hsa-miR-934
1197



hsa-miR-935
926



hsa-miR-936
0



hsa-miR-937
6



hsa-miR-938
0



hsa-miR-939
13



hsa-miR-940
71



hsa-miR-941
2312



hsa-miR-942
172



hsa-miR-943
3



hsa-miR-944
3294



hsa-miR-95
47301



hsa-miR-96
3084



hsa-miR-96*
2



hsa-miR-98
43595



hsa-miR-99a
314406



hsa-miR-99a*
1328



hsa-miR-99b
212576



hsa-miR-99b*
900

## Claims

1. A method of preparing a target nucleic acid for sequencing comprising the steps of:
a) ligating a 3′ adaptor to the target nucleic acid, wherein the 3′ adaptor consists of the nucleotide sequence of SEQ ID NO:66 or is an extended 3′ adaptor comprising the nucleotide sequence of SEQ ID NO:66 with one or more nucleotides added to the 5′ end;
b) ligating a 5′ adaptor to the 3′ adaptor-target nucleic acid complex of step (a), wherein the 5′ adaptor consists of the nucleotide sequence of SEQ ID NO:1 or is an extended 5′ adaptor comprising the nucleotide sequence of SEQ ID NO:1 with one or more nucleotides added to the 3′ end;
c) reverse transcribing the 3′ adaptor-target nucleic acid-5′ adaptor ligation product of step (b);
d) PCR amplifying the reverse transcribed ligation product of step (c); and
e) gel purifying the amplified target nucleic acid;
provided that at least one of steps (a) and (b) must utilize an extended 3′ or 5′ adaptor, respectively.

2. The method of claim 1, further comprising:
f) sequencing the gel purified target nucleic acid of step (e).

3. The method of claim 2, wherein more than one 5′ adaptor is utilized for step (b).

4. The method of claim 3, wherein step (b) utilizes a pool of 5′ adaptors, said pool comprising 16 or more different nucleotide sequences each selected from the group consisting of SEQ ID NOs:2 to 65.

5. The method of claim 1, wherein more than one 3′ adaptor is utilized for step (a).

6. The method of claim 1, wherein the target nucleic acid is a DNA or RNA molecule.

7. The method of claim 6, wherein the target nucleic acid is a smRNA.