Whole-Genome Sequencing: Automated, Nonindexed Library Preparation

2016 ◽  
Vol 2017 (3) ◽  
pp. pdb.prot094623 ◽  
Author(s):  
Elaine Mardis ◽  
W. Richard McCombie
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Library Preparation

scholarly journals A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathy E. Raven ◽  
Sophia T. Girgis ◽  
Asha Akram ◽  
Beth Blane ◽  
Danielle Leek ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
Simultaneous Processing ◽  
Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

2022 ◽  
Author(s):  
Jason Nguyen ◽  
Rebecca Hickman ◽  
Tracy Lee ◽  
Natalie Prystajecky ◽  
John Tyson
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Library Preparation ◽  
Dna Libraries ◽  
Reaction Volumes ◽  
Single Size ◽  
Stop Buffer

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed. This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e

scholarly journals Advanced Whole Genome Sequencing Using an Entirely PCR-free Massively Parallel Sequencing Workflow

2019 ◽  
Author(s):  
Hanjie Shen ◽  
Pengjuan Liu ◽  
Zhanqing Li ◽  
Fang Chen ◽  
Hui Jiang ◽  
...  
Keyword(s):  
Machine Learning ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Massively Parallel Sequencing ◽  
Variant Calling ◽  
Systematic Errors ◽  
Massively Parallel ◽  
Whole Genome ◽  
Library Preparation ◽  
Parallel Sequencing

AbstractBackgroundSystematic errors can be introduced from DNA amplification during massively parallel sequencing (MPS) library preparation and sequencing array formation. Polymerase chain reaction (PCR)-free genomic library preparation methods were previously shown to improve whole genome sequencing (WGS) quality on the Illumina platform, especially in calling insertions and deletions (InDels). We hypothesized that substantial InDel errors continue to be introduced by the remaining PCR step of DNA cluster generation. In addition to library preparation and sequencing, data analysis methods are also important for the accuracy of the output data.In recent years, several machine learning variant calling pipelines have emerged, which can correct the systematic errors from MPS and improve the data performance of variant calling.ResultsHere, PCR-free libraries were sequenced on the PCR-free DNBSEQ™ arrays from MGI Tech Co., Ltd. (referred to as MGI) to accomplish the first true PCR-free WGS which the whole process is truly not only PCR-free during library preparation but also PCR-free during sequencing. We demonstrated that PCR-based WGS libraries have significantly (about 5 times) more InDel errors than PCR-free libraries.Furthermore, PCR-free WGS libraries sequenced on the PCR-free DNBSEQ™ platform have up to 55% less InDel errors compared to the NovaSeq platform, confirming that DNA clusters contain PCR-generated errors.In addition, low coverage bias and less than 1% read duplication rate was reproducibly obtained in DNBSEQ™ PCR-free using either ultrasonic or enzymatic DNA fragmentation MGI kits combined with MGISEQ-2000. Meanwhile, variant calling performance (single-nucleotide polymorphisms (SNPs) F-score>99.94%, InDels F-score>99.6%) exceeded widely accepted standards using machine learning (ML) methods (DeepVariant or DNAscope).ConclusionsEnabled by the new PCR-free library preparation kits, ultra high-thoughput PCR-free sequencers and ML-based variant calling, true PCR-free DNBSEQ™ WGS provides a powerful solution for improving WGS accuracy while reducing cost and analysis time, thus facilitating future precision medicine, cohort studies, and large population genome projects.

scholarly journals Assessment of Ion S5 NGS protocol for SARS-CoV-2 genome sequencing

2021 ◽  
Vol 5 (2) ◽  
pp. 24
Author(s):  
Dino Pećar ◽  
Ivana Čeko ◽  
Lana Salihefendić ◽  
Rijad Konjhodžić
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Magnetic Beads ◽  
Rna Isolation ◽  
Whole Genome ◽  
Library Preparation ◽  
Rt Pcr ◽  
Sequence Coverage ◽  
Cdna Synthesis ◽  
Good Laboratory

Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.

scholarly journals Museomics of a rare taxon: placing Whalleyanidae in the Lepidoptera Tree of Life

2020 ◽  
Author(s):  
Victoria G. Twort ◽  
Joël Minet ◽  
Christopher W. Wheat ◽  
Niklas Wahlberg
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
De Novo ◽  
Sister Group ◽  
Sister Group Relationship ◽  
Whole Genome ◽  
Museum Specimens ◽  
Library Preparation ◽  
Natural History Museums ◽  
History Museums

AbstractMuseomics is a valuable tool that utilises the diverse biobanks that are natural history museums. The ability to sequence genomes from old specimens has expanded not only the variety of interesting taxa available to study but also the scope of questions that can be investigated in order to further knowledge about biodiversity. Here we present whole genome sequencing results from the enigmatic genus Whalleyana, as well as the families Callidulidae and Hyblaeidae. Library preparation was carried out on four museum specimens and one existing DNA extract and sequenced with Illumina short reads. De novo assembly resulted in highly fragmented genomes with the N50 ranging from 317 – 2,078 bp. Mining of a manually curated gene set of 332 genes from these draft genomes had an overall gene recovery rate of 64 – 90%. Phylogenetic analysis places Whalleyana as sister to Callidulidae, and Hyblaea as sister to Pyraloidea. Since the former sister-group relationship turns out to be also supported by ten morphological synapomorphies, we propose to formally assign the Whalleyanidae to the superfamily Calliduloidea. These results highlight the usefulness of not only museum specimens, but also existing DNA extracts, for whole genome sequencing and gene mining for phylogenomic studies.

SeqPlus sequencing methodology enables robust whole-genome sequencing, true variant detection, and novel genomic insights from archival esophageal carcinoma FFPE samples.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13016-e13016
Author(s):  
Shannon Terrell Bailey ◽  
Belynda Hicks ◽  
Bin Zhu ◽  
Nan Hu ◽  
Phil R. Taylor ◽  
...  
Keyword(s):  
Esophageal Carcinoma ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Limit Of Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
High Quality ◽  
Primary Tumors ◽  
Ffpe Samples ◽  
Fresh Frozen

e13016 Background: Whole-genome sequencing (WGS) of formalin-fixed, paraffin-embedded (FFPE) samples could enable novel insights from archival sample collections, yet robust FFPE WGS is challenged by fragmented DNA, uneven genomic coverage & sequencing artifacts attributed to FFPE fixation. We report our proprietary extraction & library preparation methodology (SeqPlus) with high quality, uniform WGS sequencing performance comparable to that from fresh-frozen samples. Methods: We analyzed 20 paired esophageal carcinoma (EC) samples i.e., primary tumors & matched germline samples to assess SeqPlus performance on 10-15-year-old FFPE tissues, measure variant concordance between WGS and a high-depth sequencing panel (269 genes, 400x coverage) & identify novel genomic features. Results: At a targeted 70x WGS tumor sequencing depth, 93% of the genome was covered by ³ 20 reads, 99% of bases had 10x coverage & average duplicate reads were 31%. We noted similar transition/transversion ratios & mutational spectra as from fresh-frozen EC specimens, suggesting that extraction & library preparation contributes to prior FFPE artifacts. Concordance of tumor-specific SNVs & indels derived from WGS & targeted panel was high at 86%. All 76 targeted panel-detected variants above the WGS limit of detection (mutant allele frequency [MAF] > 10%) were detected by WGS, 2 variants (2 tumors) were detected only by WGS, and 12 variants at MAF ≤ 6% (9 tumors) were only detected by the targeted panel. Tumor WGS yielded SNV, indels & CNV findings beyond variants detected by targeted sequencing. WGS enabled detection of 10.4 putative cancer variants per tumor compared to 12 variants per patient from frozen specimens and a median of 7 (up to 16) cancer-associated variants in genes outside the targeted panel. WGS copy number analysis revealed CCND1, EGFR, TP63, and SOX2amplification, CDKN2A/B deletion and additional unrecognized genomic aberrations. Conclusions: Our study reinforces the utility of high-quality, uniform WGS sequencing of archival FFPE cancer samples with SeqPlus and unlocks the potential for massive-scale retrospective genomic analysis of archived pathology samples with associated clinical & outcomes data.

scholarly journals Evaluation of Rapid Library Preparation Protocols for Whole Genome Sequencing Based Outbreak Investigation

2019 ◽  
Vol 7 ◽  
Author(s):  
Helena M. B. Seth-Smith ◽  
Ferdinando Bonfiglio ◽  
Aline Cuénod ◽  
Josiane Reist ◽  
Adrian Egli ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Outbreak Investigation ◽  
Whole Genome ◽  
Library Preparation

Automated library preparation for whole genome sequencing by centrifugal microfluidics

2021 ◽  
pp. 338954
Author(s):  
J.F. Hess ◽  
M.E. Hess ◽  
R. Zengerle ◽  
N. Paust ◽  
M. Boerries ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Centrifugal Microfluidics ◽  
Whole Genome ◽  
Library Preparation

scholarly journals Performance characterization of PCR-free whole genome sequencing for clinical diagnosis

2020 ◽  
Author(s):  
Ningzhi Zhang ◽  
Meizhen Zhou ◽  
Fanwei Zeng ◽  
Xiaodan Wang ◽  
Fengxia Liu ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Clinical Diagnosis ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Library Preparation ◽  
Experimental Parameters ◽  
Variant Detection ◽  
Disease Related Genes ◽  
Library Preparation Protocol

AbstractPurposeTo evaluate the performance of PCR-free whole genome sequencing (WGS) for clinical diagnosis, and thereby revealing how experimental parameters affect variant detection.MethodsAll the 5 NA12878 samples were sequenced using MGISEQ-2000. NA12878 samples underwent WGS with differing DNA input and library preparation protocol (PCR-based versus PCR-free protocols for library preparation). The DP (depth of coverage) and GQ (genotype quality) of each sample were compared. We developed a systematic WGS pipeline for the analysis of down-sampling samples of the 5 NA12878 samples. The performance of each sample was measured for sensitivity, coverage of depth and breadth of coverage of disease-related genes and CNVs.ResultsIn general, NA12878-2 (PCR-free WGS) showed better DP and GQ distribution than NA12878-1 (PCR-based WGS). With a mean depth of ~40X, the sensitivity of homozyous and heterozygous SNPs of NA12878-2 showed higher sensitivity (>99.77% and > 99.82%) than NA12878-1, and positive predictive value (PPV) exceeded 99.98% and 99.07%. The sensitivity and PPV of homozygous and heterozygous indels for NA12878-2 (PCR-free WGS) showed great improvement than NA128878-1. The breadths of coverage for disease-related genes and CNVs are slightly better for samples with PCR-free library preparation protocol than the sample with PCR-based library preparation protocol. DNA input also influences the performance of variant detection in samples with PCR-free WGS.ConclusionDifferent experimental parameters may affect variant detection for clinical WGS. Clinical scientists should know the range of sensitivity of variants for different methods of WGS, which would be useful when interpreting and delivering clinical reports.

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