Next-generation sequencing library preparation: simultaneous fragmentation and tagging using in vitro transposition

Syed, Fraz; Grunenwald, Haiying; Caruccio, Nicholas

doi:10.1038/nmeth.f.272

Cited by 18 publications

(16 citation statements)

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“…The Nextera transposome technology allows simultaneous fragmentation and tagmentation by using an adapted in vitro transposition. This method requires less input DNA and offers shorter preparation times [ 3 ]. The transposome consist of the transposase and a transposon complex with engineered transposon ends.…”

Section: Introductionmentioning

confidence: 99%

Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data

et al. 2016

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BackgroundIllumina’s sequencing platforms are currently the most utilised sequencing systems worldwide. The technology has rapidly evolved over recent years and provides high throughput at low costs with increasing read-lengths and true paired-end reads. However, data from any sequencing technology contains noise and our understanding of the peculiarities and sequencing errors encountered in Illumina data has lagged behind this rapid development.ResultsWe conducted a systematic investigation of errors and biases in Illumina data based on the largest collection of in vitro metagenomic data sets to date. We evaluated the Genome Analyzer II, HiSeq and MiSeq and tested state-of-the-art low input library preparation methods. Analysing in vitro metagenomic sequencing data allowed us to determine biases directly associated with the actual sequencing process. The position- and nucleotide-specific analysis revealed a substantial bias related to motifs (3mers preceding errors) ending in “GG”. On average the top three motifs were linked to 16 % of all substitution errors. Furthermore, a preferential incorporation of ddGTPs was recorded. We hypothesise that all of these biases are related to the engineered polymerase and ddNTPs which are intrinsic to any sequencing-by-synthesis method. We show that quality-score-based error removal strategies can on average remove 69 % of the substitution errors - however, the motif-bias remains.ConclusionSingle-nucleotide polymorphism changes in bacterial genomes can cause significant changes in phenotype, including antibiotic resistance and virulence, detecting them within metagenomes is therefore vital. Current error removal techniques are not designed to target the peculiarities encountered in Illumina sequencing data and other sequencing-by-synthesis methods, causing biases to persist and potentially affect any conclusions drawn from the data. In order to develop effective diagnostic and therapeutic approaches we need to be able to identify systematic sequencing errors and distinguish these errors from true genetic variation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-0976-y) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data

et al. 2016

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“…The recent development of the Nextera™ technology substantially speeds up Illumina library creation and reduces input DNA requirements down to 1 ng (Nextera-XT). In this approach DNA is simultaneously fragmented and tagged (“tagmentation”) using in vitro transposition ( Syed, Grunenwald & Caruccio, 2009 ; Caruccio, 2011 ). The resulting tagged fragments undergo a 12-cycle PCR reaction to add sequencing adaptors and sample-specific barcodes, which facilitate sample multiplexing.…”

Section: Introductionmentioning

confidence: 99%

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

Rinke

Low

Woodcroft

et al. 2016

PeerJ

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High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.

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“…However, additional amplification cycles can skew sequencing results [4], and these methods are inherently time-consuming, involving several additional enzymatic steps. Other strategies for library construction from small amounts of DNA are not suitable for ChIP analysis because they require unfragmented genomic DNA as input material [5-7]. …”

Section: Introductionmentioning

confidence: 99%

Multiplexed Illumina sequencing libraries from picogram quantities of DNA

et al. 2013

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BackgroundHigh throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps.ResultsWe report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment.ConclusionsApplication of this method allows whole genome studies from samples where material or yields are limiting.

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Next-generation sequencing library preparation: simultaneous fragmentation and tagging using in vitro transposition

Cited by 18 publications

References 0 publications

Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data

Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

Multiplexed Illumina sequencing libraries from picogram quantities of DNA

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