Rumeysa Akinci-Tolun, Isabell Haupt, Ioanna Andreou, Peter Hahn, Christian Korfhage and Nan FangQIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany

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Comparison of Different NGS Library Construction Methods for Single-Cell Sequencing

Sample to Insight

AbstractRecent advances in whole genome amplification (WGA), whole transcriptome amplification (WTA) technologies and next-

generation sequencing (NGS) have enabled whole genome or transcriptome sequencing at the single-cell level. Single-cell

sequencing studies have yielded new insights into the heterogeneity of the genome and transcriptome in individual cells. Such

heterogeneity at the single-cell level has been shown to be closely related to cellular function, differentiation, development,

and diseases.

A critical element of the single-cell sequencing workflow is sequencing library construction following WGA or WTA. An

efficient library construction method is required to convert a high percentage of the DNA fragments to an adaptor-ligated

sequencing library and to ensure high sequence complexity of the library. Furthermore, uniform representation of all genomic

regions in a sequencing library is essential for retaining all important sequence information.

Here we compared 2 library construction methods following a REPLI-g MDA-mediated WGA or WTA:

• A ligation-based library construction method using a GeneRead™ Library Prep Kit (QIAGEN)

• A ‘tagmentation’-based method using Nextera DNA Sample Prep Kit (Illumina), which simultaneously fragments and tags

DNA.

Our results demonstrated that the Nextera library construction method can be directly used with the REPLI-g-amplified DNA

following MDA reaction, without the need for DNA purification. This could be beneficial if working with a high number of

samples or if the complete workflow of single WGA/WTA and library construction should be automated. However, compared

with the tagmentation method, the ligation-based library construction method is more flexible with regard to the input DNA

amount and delivers sequencing libraries with higher complexity and less bias. This is critical for sensitive applications, such

as identification of genomic variants or comprehensive profiling of transcriptomes.

Following MDA-based WGA or WTA from 1–1000 cells, the amplified genomic DNA or cDNA, respectively, can be

subject to library construction using either a PCR-free, adaptor-ligation–based library prep method (GeneRead) or

tagmentation-based library prep method (Nextera). The workflows show the steps involved in sequencing library

construction. Both GeneRead and Nextera protocols can be completed in under 2 hours. The GeneRead library construction

protocol combines all enzymatic reactions (end-repair, A-addition, and ligation) in one reaction tube and does not require

PCR-based library amplification. The Nextera method requires an intermediate cleanup step between tagmentation and

PCR-amplification, but does not require DNA fragmentation.

GeneRead and Nextera Library Prep Workflow for Single-Cell Sequencing

Single-Cell Sequencing Library QualificationThe genome from Bacillus subtilis cells and transcriptome from HeLa S3 cells were amplified using the REPLI-g Single Cell

WGA Kit or REPLI-g Single Cell WTA Kit (both QIAGEN), respectively. The amplified genomic DNA (gDNA) or cDNA

was constructed into sequencing libraries for Illumina sequencing platforms using either the GeneRead Library Prep Kit

(QIAGEN) or Nextera DNA Library Prep Kit (Illumina). The size distribution of the sequencing libraries was analyzed on an

Agilent BioAnalyzer. GeneRead libraries from A REPLI-g–amplified gDNA and B REPLI-g-amplified cDNA. Nextera libraries

from C REPLI-g-amplified gDNA and D REPLI-g-amplified cDNA.

Sequencing libraries constructed using GeneRead had a peak size ~500 bp; Nextera libraries had a peak size above

700 bp.

Sequencing Data Quality All 4 sequencing libraries were sequenced on an Illumina MiSeq® instrument with a MiSeq Reagent Kits v2 (300-cycle,

paired end-sequencing, 2X151). FastQC software (Babraham Bioinformatics) was used to analyze the sequencing data

quality. As shown in the per-base-GC-content (1A-1D) and per-base-sequence-content (2A–2D), the Nextera Libraries had

characteristic GC bias in the first 20 nucleotides (C and D). This is likely caused by the bias of the Nextera transposase

in recognizing genome sequences for tagmentation. GeneRead libraries from A REPLI-g-amplified gDNA and B REPLI-g-

amplified cDNA. Nextera libraries from C REPLI-g-amplified gDNA and D REPLI-g-amplified cDNA. The reverse reads of

the same libraries showed same patterns in per-base-GC content as the forward reads.

GeneRead library construction workflow.

Nextera library construction workflow.

Comparison of Library Quality MetricsTo further evaluate the quality of the GeneRead and Nextera

sequencing libraries, we analyzed the following metrics:

percentages of the WGA-gDNA reads mapped to the

B. subtilis reference genome, and WGA-gDNA reads with

PHRED score of ≥30; GC bias metrics, plotted as normalized

sequence coverage on genome regions with different GC

contents; and distribution of reads for different RNA species

for the in the WTA-cDNA libraries.

SummaryBoth GeneRead and Nextera library prep methods:

• Are compatible with REPLI-g WGA and WTA for single cell sequencing

• Provide streamlined protocols to construct libraries in under 2 hours

• Deliver high library quality for sequencing MDA-amplified gDNA and cDNA

GeneRead library prep:

• PCR-free library prep for minimal bias

• High coverage uniformity

• Requires additional DNA fragmentation

Nextera library prep:

• Combined fragmentation and adaptor ligation suitable for automation

• Requires PCR amplification

• GC bias with low coverage of AT-rich region

• Bias at the ends of sequencing reads

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

Trademarks: QIAGEN®, Sample to Insight®, GeneRead™, REPLI-g® (QIAGEN Group); Illumina®, MiSeq® (Illumina, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. © 2015 QIAGEN, all rights reserved.

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Normalized coverage

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0.4

0 10 20 30 40 50 60 70 800

NexteraGeneRead

GC Contents, %

100

80

60

40

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Pecentage ofmapped ready

Pecentage of mapped readywith PHRED score ≥30

0

% GeneRead Nextera

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80

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GeneRead Nextera0

%

snRNA_pseudogenesnoRNAsnRNAscRNA_pseudogenerRNA_pseudogenepseudogenemisc_RNAmiRNAlincRNAMt_tRNA_pseudogene

1–1000 cells

WGA or WTA

Tag- mentation Cleanup PCR

amplification

Cleanup/ Adaptor removal

Sequencing library

1–1000 cells

WGA or WTA

DNA fragmenta-

tionEnd-repair A-addition Adapter

ligation

Cleanup/ Adaptor removal

Sequencing library

One tube

Tube 1 Tube 2

RNA-seq libraries made with WTA-cDNA in combination with either GeneRead or Nextera kits, showed a high percentage of reads mapped to the protein-coding genes.

Normalized coverage across genomic regions with different GC contents with WGA-gDNA libraries generated with either GeneRead or Nextera library prep kits.

Sequencing libraries constructed with both GeneRead and Nextera methods had almost 100% reads successfully mapped to the reference genome, as well as a high percentage of bases with a PHRED score of ≥30.

C D

1A

2A 2B 2C 2D

1B 1C 1D

C