APPLICATION NOTE No. 375 I December 2016

Eppendorf twin.tec® PCR Plates 96 LoBind
Increase Yield of Transcript Species andNumber of Reads of NGS Libraries
Hanae A. Henke¹, Björn Rotter²¹Eppendorf AG, Hamburg, Germany; ²GenXpro GmbH, Frankfurt am Main, Germany

Abstract
Sequencing is an increasingly important technique inlife sciences. Multiple sequencing strategies exist, but
most of them require high sample quality and, often,a large amount of starting material. Currently the mostsophisticated method is Next Generation Sequencing(NGS).Due to its complexity, many factors may interferewith the procedure and impair the quality as well as
quantity of the final library. Amongst the multitude ofthese factors, consumable selection is often overlooked.
We compared Eppendorf twin.tec PCR plate LoBind 96(Eppendorf LoBind®) with low binding characteristics andEppendorf twin.tec PCR plate 96 (standard) for theirsequencing read depth and number of transcript species.
To demonstrate the advantage of Eppendorf LoBind
consumables, two NGS libraries (10 and 50 ng total RNAinput) were prepared in both plate types, sequenced,and the data were compared. It was shown that samples
processed in Eppendorf LoBind plates produced a higher
average number of reads, as well as broader variation oftranscript species. NGS libraries of low quality and low
starting material should therefore benefit even more fromusing the Eppendorf LoBind PCR plates.

Introduction
Nowadays, sequencing is one of the most important technologies in almost every research field. A number of approaches
exist and the classic Sanger chain termination method invented in 1977 was further developed over the past decades.
Amongst these methods, NGS is the most sophisticated,which offers the possibility to sequence up to 1.8 terabasesin one sequencing run [1]. Preparing a library for NGS canbe a long, multi-step process. Typically, four major steps areinvolved. First, the DNA sample is fragmented. Secondly,
the broken DNA ends are enzymatically repaired for furthermodification.

The third step includes attaching oligonucleotides to bothends of the fragments to allow sample distinction and universal amplification. The fourth step is the enrichment PCR
to increase the amount of the library. Quality of a library
can be assessed by many metrics after a sequencing run. A
commonly-used indice is the number of reads, which should
be as high as possible (with controlled cluster density) to increase the chance of capturing all variants. Another indice isthe number of various transcript species (in the case of RNA
sequencing), which can be used to gauge the faithfulness ofsequencing data.

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Another important element for successful sequencing issample quality. Ideally samples are supplied at high purity
and quantity. But in reality, samples derived from ending sources such as FFPE or forensic samples can be very
limited and poor in quality. One of the methods to treat
these samples is Massive Analysis of cDNA Ends (MACE)[2]. In MACE, only one read per transcript molecule issequenced. So short and rare transcripts can be identified
at 10-20 times lower sequencing depth when compared tofull length RNA-Seq. Moreover, no length-based normalization is required [3]. Therefore, this technique can be used
to process challenging samples for NGS sequencing. Theanalysis method TrueQuant offers the possibility to cleansequencing data from sequencing artifacts and low quality
reads. When it comes to sensitive samples with low input,
high-quality cluster generation and good sequencing depth
and coverage are essential to data fidelity. So improving thequality of critical aspects, including purity, number of reads,
and preservation of transcript variants can all lead to better
sequencing results.

One way to ensure high sample quality of a NGS library isusing special consumables that protect the DNA. Multiple
studies have found that standard laboratory polypropyleneconsumables may lead to DNA denaturation, multimerization, as well as increased binding of short DNA fragments tothe plastic surface [4, 5]. Additionally, substances leachedfrom the plastics can interfere with the sample and inhibit
binding of receptors, increase photometric readings, andalter the activity of enzymes [6]. Using plastics that do notaffect DNA, such as polyallomers, for the production oflaboratory consumables is one approach to answer thosechallenges [4]. To demonstrate the effectiveness of thelow-binding feature for NGS workflows, we used Eppendorftwin.tec PCR plate 96 LoBind (Eppendorf LoBind) withlow binding characteristics and Eppendorf twin.tec PCR plate
96 (standard) for the preparation of NGS libraries. Thenwe compared their performance by number of reads fromsequencing, and variation of transcript species.

Materials and Methods
Comparison of qPCR efficacy in Eppendorf LoBind andstandard PCR plates
A general comparison of qPCR efficiency between Eppendorf
twin.tec PCR plate 96 LoBind, semi-skirted (EppendorfLoBind; Lot. No: E161876M; Eppendorf AG, Hamburg,
Germany) and Eppendorf twin.tec PCR plate 96, semiskirted (standard; Eppendorf AG, Hamburg, Germany)was done to ensure that the LoBind material does notinterfere with the PCR reaction. HOT MOLPol EvaGreen®
qPCR Mix Plus (projodis GmbH, Butzbach, Germany) wasused in 20 µL reaction volume. The template used was
1 µL of a pooled NGS library (0.1 ng/uL) produced andpurified via SPRIselect® SPRI bead-based size selection(Beckman Coulter, California, USA). The reactions wereprepared in both the Eppendorf LoBind and standard
plates, then consolidated to a MicroAmp® Fast Optical 48well reaction plate (REF: 4375816, Applied Biosystems®,
California, USA) for qPCR analysis. The cycler used wasApplied Bio-systems StepOne™ Real-Time PCR System(Applied Bio-systems, California, USA).

Comparison of sequencing efficiency of NGS library ofprimary cells in Eppendorf LoBind and standard PCR plates
10 and 50 ng total RNA from primary HUVEC cells wasused to prepare a NGS library according to the MACE
protocol [2] by GenXpro GmbH, Frankfurt a. M., Germany.
cDNA synthesis and all following steps were accomplishedin Eppendorf LoBind and standard plates. Both plate types
were treated in parallel with 3 replicates each. Ten PCR
cycles were applied for both groups. Sequencing of all products was done on the same Illumina® NextSeq® 500 lane
(Illumina, Inc., California, USA). The adapter sequences had
a length of 75 bps. All sequencing data was cleared fromartifacts and low quality reads with the TrueQuant method(GenXpro GmbH, Frankfurt a. M., Germany). All MACE
sequencing data was annotated on the human genome
hg19 using the GenXpro annotation pipeline with novoalign(http://novocraft.com/). All annotation coordinates werealigned with Refseq-tracks to receive the genetic information. Normalization of the data was accomplished by calculating the number of each transcript over the number of all
sequenced molecules followed by multiplication of106 (TPM = tags per million).

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Results and Discussion
Comparison of qPCR efficacy in Eppendorf LoBind andstandard PCR plates
To demonstrate that the Eppendorf LoBind property does
not affect the PCR performance, amplification rate in theEppendorf LoBind and standard PCR plates must be comparable. A qPCR experiment in both plate types was doneusing the same NGS library as template. Figure 1 shows thatthe amplification rates of a target in the Eppendorf LoBind
and standard plates are similar, at Ct values of 8.83 and 8.81,
respectively.

qPCR efficacy in LoBind and standard plates

Eppendorf LoBind

standard

Figure 1: Comparison of amplification rates and PCR efficacy in
Eppendorf LoBind and standard plates (n=3).

Comparison of sequencing efficiency of a NGS library ofprimary cells in Eppendorf LoBind and standard PCR plates
To compare the effectiveness of sequencing in Eppendorf
LoBind and standard plates, NGS libraries were preparedfrom 10 and 50 ng total RNA. Then both libraries were sequenced on the same lane of a flow cell. All sequences werecleaned-up by the TrueQuant method. This method excludes
sequencing artifacts as well as low quality reads, resulting
in an average of 549,165 reads from the library preparedin the Eppendorf LoBind plate and 442,699 reads from thestandard plate with 10 ng total RNA input. The data showsa lead of 20 % in reads using the LoBind plates versus thestandard ones, which is repeated with 50 ng group (~23 %more reads from the Eppendorf LoBind plate). The average number of reads in LoBind plates was 2,836,281 whilein standard plates 2,184,589 reads could be detected. This
indicates that the usage of Eppendorf LoBind plates for thepreparation of NGS libraries leads to more raw data output,
leaving room for evaluation and a higher chance of catching
all desired sequences in the library.

AAverage number of reads (10 ng)

Eppendorf LoBind

Bstandard

Average number of reads (50 ng)

Eppendorf LoBindstandard

Figure 2: The average number of reads of a 10 ng (2 A) and 50 ng (2 B) NGS
library prepared from total RNA of primary HUVEC cells in Eppendorf LoBind
and standard plates. The average number of reads is up to 23 % higher in
Eppendorf LoBind than in standard plates (n=3).

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Not only the average read depth is a quality measure of asequencing run, but also the number of different transcript
species detected. The more different species identified, themore comprehensive the sequencing data is, and with thisthe evaluation possibilities increase. It was found that using
Eppendorf LoBind plates for the preparation of NGS libraries led to an increase in the average number of transcriptspecies. 13,800 different transcript species in LoBind plates
and 12,800 in standard plates from 10 ng total RNA for thelibrary set up shows that Eppendorf LoBind plates helped
to preserve the transcript variability (figure 3 A). This enhancement was more robust as the starting material increased (50 ng), an average number of 18,000 in Eppendorf
LoBind plates and 12,000 in standard plates or 30 % moretranscript species from the Eppendorf LoBind plates werereported (figure 3 B).

AAverage number of transcript species of 10 ng
total RNA library

Eppendorf LoBind

Bstandard

Average number of transcript species of 50 ng
total RNA library

Eppendorf LoBind

standard

Figure 3: The average number of transcript species of NGS libraries preparedfrom 10 ng (3 A) and 50 ng (3 B) total RNA of primary HUVEC cells in Eppendorf LoBind and standard plates. The average number of transcript species isup to 30 % higher in Eppendorf LoBind than in standard plates (n=3).

Conclusion
Preparing NGS libraries in Eppendorf LoBind plates insteadof standard plates showed multiple advantages. While thePCR efficiency in both plate types is identical, the quality
and quantity of the resulting sequencing data is improved.
Moreover, especially NGS libraries of lower quality may
benefit from using Eppendorf LoBind plates. More reads
and transcript species were identified by sequencing thesame library prepared in the LoBind plates as comparedto preparation in the standard plates. So a low quality NGS
library prepared in a Eppendorf LoBind plate can havecomparable results to a high quality library prepared in astandard plate. Using Eppendorf LoBind plates means lesswork and fewer repetitions have to be done during library
preparation. This is especially advantageous when low quality
or low starting material has to be used. An increased amountof raw sequencing data by preparing a library in Eppendorf
LoBind plates adds to more assurance at detecting rare
species from challenging samples.

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Acknowledgement
We want to thank Dr. Björn Rotter and his team at GenXpro GmbH for their enthusiasm in the project and thescientific guidance.

Literature[1] www.illumina.com/technology/next-generation-sequencing.html[2] Hofer TP, Zawad AM, Frankenberger M, Skokann K, Satzl AA, gesierich W, Schuberth M, Levin J, Danek A,Rotter B, Heine GH, Ziegler-Heitbrook L. Characterization of subsets of the CD16-positive monocytes:
impact of granulomatous inflammation and M-CSF-receptor mutation. Blood 2015 Oct 6. Pii: blood-2015-06-651331[3] http://genxpro.net/sequencing/transcriptome/mace-massive-analysis-of-cdna-ends/[4] Gaillard C, Strauss F. Avoiding adsorption of DNA to polypropylene tubes and denaturation of short DNA fragments.
Science, Technical Tips online 1998, Vol. 3: 63-65[5] Belotserkovskii BP. Polypropylene Tube Surfaces May Induce Denaturation and Multimerization of DNA.
Science, Technical comments 1996, Vol. 271: 222-223[6] Grzeskowiak R, Gerke N. Leachables: Minimizing the Influence of Plastic Consumables on the Laboratory Workflows.
Eppendorf White Paper 26; www.eppendorf.com

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Ordering information
Description

Order no. international

Order no. North America

Eppendorf twin.tec PCR plates 96 Eppendorf LoBind , semi-skirted

0030 129.504

0030129504

Eppendorf twin.tec® PCR plates 96 Eppendorf LoBind®, skirted

0030 129.512

0030129512®

®

Your local distributor: www.eppendorf.com/contact
Eppendorf AG · 22331 Hamburg · Germany
eppendorf@eppendorf.com · www.eppendorf.com

www.eppendorf.com
LabChip® is a registered trademark of Perkin Elmer, USA. SPRIselect® is a registered trademark of Beckman Coulter, Inc., USA. Illumina® and NextSeq® are registered trademarks of Illumina, Inc., USA.
EvaGreen® is a registered trademark of Biotium, Inc., USA. Applied Biosystems® and MicroAmp® are registered trademarks of Applied Biosystems LLC, USA. StepOne™ is a trademark of Applied Biosystems LLC, USA.
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