Multiplex Illumina Sequencing Using DNA Barcoding

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The amount of sequence obtained by modern sequencing machines greatly exceeds the sequencing depth requirements of many experiments, especially those involving organisms with small genomes. In the interest of economy and efficiency, various strategies have been developed for multiplexing, in which samples are uniquely tagged with short identifying sequences known as barcodes, pooled, and then sequenced together in a single lane. The resulting combined sequence data are subsequently sorted by barcode before bioinformatic analysis. This unit contains a barcoding protocol for the preparation of up to 96 ChIP samples for multiplex sequencing in a single flow cell lane on the Illumina platform. This strategy may be extended to even larger numbers of samples and may also be generalized to other sequencing applications or sequencing platforms. Curr. Protoc. Mol. Biol. 101:7.11.1?7.11.11. © 2013 by John Wiley & Sons, Inc.

Keywords: barcoding; multiplexing; deep sequencing; next?generation sequencing; ChIP?seq

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Introduction
Basic Protocol 1: Construction of Barcoded ChIP‐seq Libraries
Support Protocol 1: Preparation of Barcoded Y‐Adapters
Support Protocol 2: DNA Purification and Size Selection Using SPRI‐Magnetic Beads
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Construction of Barcoded ChIP‐seq Libraries

Materials

1 to 10 ng ChIP DNA (see unit 21.3 )
Nuclease‐free water
10× T4 DNA Ligase buffer
10 mM dNTP mix
T4 DNA polymerase, 3000 U/ml (NEB)
T4 Polynucleotide kinase, 10,000 U/ml (NEB)
DNA polymerase I, large (Klenow) fragment, 5000 U/ml (NEB)
QIAquick PCR Purification kit (QIAGEN), or equivalent, containing Elution buffer (EB)
10× NEBuffer 2 (NEB)
10 mM dATP
Klenow Fragment (3′→5′ exo‐), 5000 U/ml (NEB)
QIAquick MinElute PCR Purification kit (QIAGEN)
10 µM annealed Y‐adapters (from protocol 2 )
Quick Ligation Kit (NEB) containing:
- 2× Quick Ligation Reaction buffer
- Quick T4 DNA ligase
Phusion High‐Fidelity DNA Polymerase, 2000 U/ml (NEB) containing 5× Phusion HF Reaction buffer
10 µM PCR primer mix (PCR1 and PCR2; see Table 7.11.1 )
25 mM dNTP Mix
QIAquick Gel Extraction kit (QIAGEN)

1.5‐ml microcentrifuge tubes
37°C heat block
0.5‐ml PCR tubes
Thermal cycler

Gel electrophoresis apparatus (unit 2.5 )

Table 7.1.1 MaterialsOligonucleotide Sequences for Illumina Paired‐End Y‐Adapter and PCR Primers

Oligo	Sequence
Barcoded adapter oligo 1 (BA1)	5′ ACACTCTTTCCCTACACGACGCTCTTCCGATCT‐barcode ‐T 3′
Barcoded adapter oligo 2 (BA2)	5′ p‐barcode (reverse complement)‐AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 3′ a
PCR primer forward (PCR1)	CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATundefinedT b
PCR primer reverse (PCR2)	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATundefinedT b

^a 5′ p indicates 5′ phosphorylation.

Support Protocol 1: Preparation of Barcoded Y‐Adapters

Materials

Oligo BA1 and BA2 (see Table 7.11.1 )
Annealing buffer (see recipe )

1.5‐ml microcentrifuge tubes
0.5‐ml PCR tubes
Thermal cycler

Support Protocol 2: DNA Purification and Size Selection Using SPRI‐Magnetic Beads

Materials

DNA SizeSelector‐I beads (Aline Biosciences)
DNA sample
70% Ethanol
EB (from Qiaquick kit) or H₂ O

Vortex mixer
1.5‐ml microcentrifuge tubes
Magnetic stand

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Figures

Figure 7.11.1 Outline for preparation of 5′ barcoded DNA libraries for Illumina sequencing. DNA fragments ranging from 100 bp to 500 bp are first subjected to end‐repair, 5′ phosphorylation, and 3′ A base addition. Y‐adapters with unique barcode sequences (depicted as blue lines) are then ligated onto both ends of DNA fragments. Purified ligation products are subjected to PCR amplification with primers (PCR1/2) containing the full sequences required for hybridization to the Illumina sequencing flow cell. The quality and quantity of purified library DNA can be evaluated by qPCR and Bioanalyzer analyses, respectively. DNA libraries are then ready for mixing and Illumina sequencing. When mixing libraries with severe primer dimer contamination, several rounds of gel or SPRI purifications may be needed to completely eliminate the contaminating primer dimers. A Bioanalyzer assay may be used to check for the presence of primer dimers in the final pooled library.

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Literature Cited

	Church, G.M. and Kieffer‐Higgins, S. 1988. Multiplex DNA sequencing. Science 240:185‐188.
	Lefrançois, P., Euskirchen, G.M., Auerbach, R.K., Rozowsky, J., Gibson, T., Yellman, C.M., Gerstein, M., and Snyder, M. 2009. Efficient yeast ChIP‐Seq using multiplex short‐read DNA sequencing. BMC Genomics 10:37.
	Meyer, M., Stenzel, U., Myles, S., Prüfer, K., and Hofreiter, M. 2007. Targeted high‐throughput sequencing of tagged nucleic acid samples. Nucleic Acids Res. 35:e97.
	Tirosh, I., Wong, K.H., Barkai, N., and Struhl, K. 2011. Extensive divergence of yeast stress responses through transitions between induced and constitutive activation. Proc. Natl. Acad. Sci. U.S.A. 108:16693–16698.
	Wong, K.H. and Struhl, K. 2011. The Cyc8‐Tup1 complex inhibits transcription primarily by masking the activation domain of the recruiting protein. Genes Dev. 25:2525–2539.
Internet Resource
	http://zarmik.hms.harvard.edu/Barcoding
	Python scripts for data pre‐processing and analysis.