TITLE: Using  sequins with RNA sequencing.
AUTHORS: Tim Mercer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Sequins are synthetic RNA controls that that are ‘spiked-in’ to your RNA sample, and undergo concurrent library preparation, sequencing and analysis. The sequins are then analyzed as internal controls in the output NGS library. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol describes the laboratory steps required to dilute, store and spike the sequins into your RNA sample prior to library preparation for RNA sequencing. We also describe the bioinformatic steps to analyse sequins within your read (.FASTQ) or alignment (.BAM) files.</div></div></div>

[STEPS]
?. [Laboratory Steps]
Receiving sequins.Upon receipt of RNA sequins, first check to ensure they have not thawed during shipment. Please contact us if you have any concerns. Immediately transfer the RNA sequins to frozen storage at -80°C (sequins should not be stored in a -20°C frost-free freezer). Each tube contains RNA sequins in 10 μL solution, which is typically sufficient for ~100 RNAseq libraries. On first thaw, spin the tube down to collect the contents at the bottom of tube, and prepare smaller single-use aliquots to minimize subsequent freeze-thaw cycles.
?. [Laboratory Steps]
Preparing sequin stocks. RNA sequins are provided in solution in nuclease-free water at a concentration of 15 ng/μL. Please use the table below to determine the amount and dilution of sequins that should be added to the sample RNA amount (please note users should dilute sequins even further when using with targeted RNA sequencing applications) :   ABCDEF1Sample RNA Amount.Dilution Volume (ddH20)Diluted Sequins Concentration.Sequin Volume to
Add to Sample.Mass of Sequins
Added.Final Sequin
Concentration220ng740ul0.2ng/ul1ul0.2ng1%350ng290ul0.5ng/ul1ul0.5ng1%4100ng140ul1ng/ul1ul1.0ng1%5500ng20ul5ng/ul1ul5.0ng1%61000ng6ul10ng/ul1ul10.0ng1%NOTE | RNA sequins are provided in two mixtures (A and B). Each mixture contain the same sequin transcripts, but at different molar ratios, thereby emulating fold-change differences in gene expression and alternative splicing between the two mixtures. Mixture A and B can be added to different samples to allow the detection of these known fold-differences.
ABCDEF1Sample RNA Amount.Dilution Volume (ddH20)Diluted Sequins Concentration.Sequin Volume to
Add to Sample.Mass of Sequins
Added.Final Sequin
Concentration220ng740ul0.2ng/ul1ul0.2ng1%350ng290ul0.5ng/ul1ul0.5ng1%4100ng140ul1ng/ul1ul1.0ng1%5500ng20ul5ng/ul1ul5.0ng1%61000ng6ul10ng/ul1ul10.0ng1%
?. [Laboratory Steps]
Sequencing. The library that is generated from the combined RNA sample and sequins is then sequenced according to manufacturer’s instructions.
?. [Analysis of .FASTQ libraries (Option 1).]
Analysis of .FASTQ libraries (option 1).To directly analyze the sequin from your library .FASTQ files, run the following command: anaquin rna -t 24 -o results --calibrate 0.005 -1 K562_SequinMixA.Rep1.R1.fq.gz \-2 K562_SequinMixA.Rep1.R2.fq.gzIn this example command, we used the --calibrate 0.005 option to subsample sequin reads to coprise 0.5% of total reads in the NGS library.
?. [Analysis of .BAM libraries (Option 2).]
Build index with decoy chromosome. The sequins can also be aligned to a decoy chromosome (chrQ) that is indexed along with the reference genome assembly. To build this combined index, the user first concatenates the human genome sequences to the decoy chromosomes into a single file: cat hg38.fa rnasequin_decoychr_2.4.fa >hg38_decoy.fa User then builds an index from these combined files using their alignment tool of choice. For example, using the STAR aligner (Doben et. al., 2013): mkdir /path/to/star_genome_dirSTAR --runMode genomeGenerate \--genomeDir ./star_genome_dir \--genomeFastaFiles hg38_decoy.fa
?. [Analysis of .BAM libraries (Option 2).]
Alignment (using STAR).We then align the library to the combined index:STAR --runThreadN 8 \--runMode alignReads \--genomeDir /path/to/star_genome_dir \--readFilesIn K562_SequinMixA.Rep1.R1.fq.gz K562_SequinMixA.Rep1.R2.fq.gz \ --readFilesCommand zcat \ --outSAMtype BAM SortedByCoordinate \ --outFileNamePrefix K562_SequinMixA.Rep1
?. [Analysis of .BAM libraries (Option 2).]
Analysis of sequins.We finally use anaquin rna to analyse the .BAM alignment files:anaquin rna -t 24 -o results --calibrate 0.005 --combined K562_SequinMixA.Rep1.bamIn this example command, we used the --calibrate 0.005 option to subsample sequin alignments to comprise 0.5% of total alignments from the NGS library.
?. [Output Results.]
When anaquin rna is complete, the following files are generated in the output directory: Analysis.anaquin.log – Log files recording the usage and execution of sequin processes. rna_report.html – Useful visual report describing sequin performance in library. Load with browser. rna_summary.txt - Summary statistics describing sequins and libraries. rna_sequins.tsv - Detailed statistics on each individual sequin. rna_sequins_calibrated.tsv - Detailed statistics on individual sequins following calibration. Libraries. rna_sequin_gene_table.tsv – gene-level quantification of sequins. rna_sequin_isoform_table.tsv – isoform-level quantification of sequins. rna_sample_* - Sample alignments/reads (excludes sequins). rna_sequins_* - Alignments/reads derived from sequins