Abstract 1706: High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types

2019 ◽  
Author(s):  
Shannon T. Bailey ◽  
James Lund ◽  
Muhammad Ekram ◽  
Wanfeng Yu ◽  
Richard T. Williams ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
High Quality ◽  
Ffpe Samples

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.

Abstract 427: High-quality whole-genome sequencing of FFPE samples

2018 ◽  
Author(s):  
Shannon T. Bailey ◽  
Jim Lund ◽  
Hao Wang ◽  
Weiren Cui ◽  
Chen Hao ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
High Quality ◽  
Ffpe Samples

Abstract 1706: High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types

2019 ◽  
Author(s):  
Shannon T. Bailey ◽  
James Lund ◽  
Muhammad Ekram ◽  
Wanfeng Yu ◽  
Richard T. Williams ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
High Quality ◽  
Ffpe Samples

High-quality whole-genome sequencing of FFPE samples.

2018 ◽  
Vol 36 (15_suppl) ◽  
pp. e13500-e13500 ◽  
Author(s):  
Shannon Terrell Bailey ◽  
Jim Lund ◽  
Hao Wang ◽  
Hao Chen ◽  
Hongye Sun ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
High Quality ◽  
Ffpe Samples

scholarly journals Whole-Genome Sequencing Applied to the Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli O157:H7 in Argentina: TABLE 1 

2016 ◽  
Vol 4 (6) ◽  
Author(s):  
Claudia Carolina Carbonari ◽  
Nahuel Fittipaldi ◽  
Sarah Teatero ◽  
Taryn B. T. Athey ◽  
Luis Pianciola ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Epidemiology ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Shiga Toxin ◽  
Whole Genome ◽  
Escherichia Coli O157 ◽  
Genome Sequences ◽  
High Quality ◽  
Human Infections

Shiga toxin-producing Escherichia coli strains are worldwide associated with sporadic human infections and outbreaks. In this work, we report the availability of high-quality draft whole-genome sequences for 19 O157:H7 strains isolated in Argentina.

scholarly journals Exploring the Diversity of Bacillus whole genome sequencing projects using Peasant, the Prokaryotic Assembly and Annotation Tool

2017 ◽  
Author(s):  
Jonathon Brenner ◽  
Laurynas Kalesinskas ◽  
Catherine Putonti
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Annotation Tool ◽  
Illumina Platform ◽  
Desktop Computer ◽  
High Quality ◽  
Sequencing Project ◽  
Computational Resources

ABSTRACTBackgroundThe persistent decrease in cost and difficulty of whole genome sequencing of microbial organisms has led to a dramatic increase in the number of species and strains characterized from a wide variety of environments. Microbial genome sequencing can now be conducted by small laboratories and as part of undergraduate curriculum. While sequencing is routine in microbiology, assembly, annotation and downstream analyses still require computational resources and expertise, often necessitating familiarity with programming languages. To address this problem, we have created a light-weight, user-friendly tool for the assembly and annotation of microbial sequencing projects.ResultsThe Prokaryotic Assembly and Annotation Tool, Peasant, automates the processes of read quality control, genome assembly, and annotation for microbial sequencing projects. High-quality assemblies and annotations can be generated by Peasant without the need of programming expertise or high-performance computing resources. Furthermore, statistics are calculated so that users can evaluate their sequencing project. To illustrate the computational speed and accuracy of Peasant, the SRA records of 322 Illumina platform whole genome sequencing assays for Bacillus species were retrieved from NCBI, assembled and annotated on a single desktop computer. From the assemblies and annotations produced, a comprehensive analysis of the diversity of over 200 high-quality samples was conducted, looking at both the 16S rRNA phylogenetic marker as well as the Bacillus core genome.ConclusionsPeasant provides an intuitive solution for high-quality whole genome sequence assembly and annotation for users with limited programing experience and/or computational resources. The analysis of the Bacillus whole genome sequencing projects exemplifies the utility of this tool. Furthermore, the study conducted here provides insight into the diversity of the species, the largest such comparison conducted to date.

scholarly journals Fast and inexpensive whole genome sequencing library preparation from intact yeast cells

2020 ◽  
Author(s):  
Sibylle C Vonesch ◽  
Shengdi Li ◽  
Chelsea Szu Tu ◽  
Bianca P Hennig ◽  
Nikolay Dobrev ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genomic Dna ◽  
Large Scale ◽  
Massively Parallel Sequencing ◽  
Yeast Cells ◽  
Whole Genome ◽  
High Quality ◽  
Rapid Preparation ◽  
Yeast Cultures

ABSTRACTThrough the increase in the capacity of sequencing machines massively parallel sequencing of thousands of samples in a single run is now possible. With the improved throughput and resulting drop in the price of sequencing, the cost and time for preparation of sequencing libraries have become the major bottleneck in large-scale experiments. Methods using a hyperactive variant of the Tn5 transposase efficiently generate libraries starting from cDNA or genomic DNA in a few hours and are highly scalable. For genome sequencing, however, the time and effort spent on genomic DNA isolation limits the practicability of sequencing large numbers of samples. Here, we describe a highly scalable method for preparing high quality whole-genome sequencing libraries directly from yeast cultures in less than three hours at 34 cents per sample. We skip the rate-limiting step of genomic DNA extraction by directly tagmenting yeast spheroplasts and add a nucleosome release step prior to enrichment PCR to improve the evenness of genomic coverage. Resulting libraries do not show any GC-bias and are comparable in quality to libraries processed from genomic DNA with a commercially available Tn5-based kit. We use our protocol to investigate CRISPR/Cas9 on- and off-target edits and reliably detect edited variants and shared polymorphisms between strains. Our protocol enables rapid preparation of unbiased and high-quality, sequencing-ready indexed libraries for hundreds of yeast strains in a single day at a low price. By adjusting individual steps of our workflow we expect that our protocol can be adapted to other organisms.

scholarly journals Les gènes des enfants de Tchernobyl

2021 ◽  
Vol 37 (8-9) ◽  
pp. 802-805
Author(s):  
Bertrand Jordan
Keyword(s):  
Radiation Dose ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Large Scale ◽  
Whole Genome ◽  
Transgenerational Effects ◽  
High Quality ◽  
Chernobyl Disaster ◽  
Large Scale Study ◽  
New Mutations

Transgenerational effects have long been expected in children from parents exposed to radiation from atomic bombs in Japan in 1945 or from the Chernobyl disaster in 1986. These effects have in fact proven hard to detect. A new large-scale study based on high-quality whole genome sequencing of father/mother/child trios in which the parental radiation dose is known now demonstrates that the rate of new mutations (50/70 per generation) is not detectably increased when comparing irradiated and non-irradiated parents. This solid data shows conclusively that transgenerational effects of irradiation from the Chernobyl disaster are absent or undetectable.

scholarly journals Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Annie N. Cowell ◽  
Dorothy E. Loy ◽  
Sesh A. Sundararaman ◽  
Hugo Valdivia ◽  
Kathleen Fisch ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Whole Genome Sequencing ◽  
Plasmodium Vivax ◽  
Genome Sequencing ◽  
Whole Genome Amplification ◽  
Microbial Pathogens ◽  
Clinical Samples ◽  
Whole Genome ◽  
High Quality ◽  
Genome Amplification

ABSTRACT Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax , the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. IMPORTANCE Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.

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