Optimized library preparation method for next-generation sequencing

Syed, Fraz; Grunenwald, Haiying; Caruccio, Nicholas

doi:10.1038/nmeth.f.269

Cited by 14 publications

(10 citation statements)

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“…Pooled amplicons were then amplified in the second PCR in order to add Illumina P5/P7 adaptor sequences (Syed et al . ). The pool was diluted by between 10× and 100×, depending on the density of the electrophoresis band, and PCR utilizing primers carrying P5/P7 adaptors was performed for 12 cycles (according to Illumina MiSeq protocol).…”

Section: Methodsmentioning

confidence: 97%

Testing genotyping strategies for ultra‐deep sequencing of a co‐amplifying gene family: MHC class I in a passerine bird

Biedrzycka

Sebastián

Migalska

et al. 2016

Molecular Ecology Resources

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Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage.

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Section: Methodsmentioning

confidence: 97%

Testing genotyping strategies for ultra‐deep sequencing of a co‐amplifying gene family: MHC class I in a passerine bird

Biedrzycka

Sebastián

Migalska

et al. 2016

Molecular Ecology Resources

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“…Transposase‐fragmented and bead‐based size‐selected libraries were chosen as a model in this investigation because of the relatively straightforward library preparation protocol, easily adapted for automating library preparation as previously demonstrated on our DMF platform . Instead of a standard 8–10 step Illumina library preparation protocol, Nextera technology using in vitro transposition allows a library to be prepared in three steps in less than 2 h: an enzymatic fragmentation and adaptor ligation, a sample cleanup, and a limited‐cycle PCR for tagged‐library enrichment . All Illumina‐compatible library samples were analyzed without dilutions and 50 pg/μL DNA ladder serving as an internal standard was included in each library sample droplet for accurate determination of size‐distribution and quantification of the libraries.…”

Section: Resultsmentioning

confidence: 99%

Quality control of next‐generation sequencing library through an integrative digital microfluidic platform

et al. 2012

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We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/μL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments.

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“…Classically, this A-addition stage is catalysed by Klenow Fragment (minus 3' to 5' exonuclease) or other polymerases with terminal transferase activity [40]. Ligase repaired library fragments, followed by reaction cleanup and DNA size selection to remove free library adapters [41]. The approaches for size selection of library comprise agrose gel separation, the usage of magnetic beads, or progressive column-based refinement methods [42].…”

Section: Library Preparationmentioning

confidence: 99%

“…The sequencing occurs from beginning to end a cycle of washing and flooding the fragments with the recognized nucleotides in a sequential order [57]. As nucleotides incorporate into the growing DNA strand, they are digitally recorded as sequence [58]. The PGM and the MiSeq each rely on a slightly different mechanism for detecting nucleotide sequence information [59].…”

Section: Sequencingmentioning

confidence: 99%

Next-generation sequencing technologies as emergent tools and their challenges in viral diagnostic

Singh¹

2018

biomedicalresearch

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Next-Generation Sequencing (NGS) has the range of high-throughput quickly adapted into the countless facet of viral diagnostic research. This method has been extensively applying in full genome sequencing, genetic diversity identification, transcriptomic routine diagnostic work patient care and management, and the new understanding of the interactions between viral and host transcriptome, to promote virus research. An exciting era of viral exploration has begun and will set us new challenges to understand the role of newly discovered viral diversity in both disease and health. In this review, discuss about the preparation of viral nucleic acid templates for sequencing, assay formats underlying next-generation sequencing systems, methods for imaging and base calling, quality control, data processing pipelines and bioinformatics approaches for sequence alignment, variant calling and suggestions for selecting suitable tools. Also discuss of the most important advances that the new sequencing technologies have brought to the fields of clinical virology and challenges behind it.

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Optimized library preparation method for next-generation sequencing

Cited by 14 publications

References 0 publications

Testing genotyping strategies for ultra‐deep sequencing of a co‐amplifying gene family: MHC class I in a passerine bird

Testing genotyping strategies for ultra‐deep sequencing of a co‐amplifying gene family: MHC class I in a passerine bird

Quality control of next‐generation sequencing library through an integrative digital microfluidic platform

Next-generation sequencing technologies as emergent tools and their challenges in viral diagnostic

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