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

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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Faculty Opinions recommendation of Characterization of uterine leiomyomas by whole-genome sequencing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718021966.793479049 ◽

2013 ◽

Author(s):

Hiroshi Ishikawa

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Uterine Leiomyomas ◽

Whole Genome

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Characterization of Shiga Toxin‐Producing Escherichia coli Isolated From Cattle and Sheep in Xinjiang Province, China, Using Whole‐Genome Sequencing

Transboundary and Emerging Diseases ◽

10.1111/tbed.13999 ◽

2021 ◽

Author(s):

Yingyu Liu ◽

Huoming Li ◽

Xuhua Chen ◽

Panpan Tong ◽

Yan Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Shiga Toxin ◽

Whole Genome ◽

Cattle And Sheep

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

Scientific Reports ◽

10.1038/s41598-020-80031-8 ◽

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.

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SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

Scientific Reports ◽

10.1038/s41598-019-55144-4 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Weili Cai ◽

Schyler Nunziata ◽

John Rascoe ◽

Michael J. Stulberg

Keyword(s):

Whole Genome Sequencing ◽

Molecular Characterization ◽

Genome Sequencing ◽

Whole Genome Sequence ◽

Whole Genome ◽

Genome Coverage ◽

Metagenomic Sample ◽

Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

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

10.17504/protocols.io.b3vgqn3w ◽

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

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First isolation and characterization of Brucella suis from yak

Genome ◽

10.1139/gen-2019-0101 ◽

2020 ◽

Vol 63 (8) ◽

pp. 397-405

Author(s):

Xiaowen Yang ◽

Ning Wang ◽

Xiaofang Cao ◽

Pengfei Bie ◽

Zhifeng Xing ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Health And Safety ◽

Host Cells ◽

Whole Genome ◽

Genetic Characteristics ◽

Qinghai Province ◽

Isolation And Characterization ◽

Smooth Strain

Brucella spp., facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonosis, affect public health and safety. A Brucella strain was isolated from yak in Qinghai Province. To detect whether this isolate could cause an outbreak of brucellosis and to reveal its genetic characteristics, several typing and whole-genome sequencing methods were applied to identify its species and genetic characteristics. Phylogenetic analysis based on MLVA and whole-genome sequencing revealed the genetic characteristics of the isolated strain. The results showed that the isolated strain is a B. suis biovar 1 smooth strain, and this isolate was named B. suis QH05. The results of comparative genomics and MLVA showed that B. suis QH05 is not a vaccine strain. Comparison with other B. suis strains isolated from humans and animals indicated that B. suis QH05 may be linked to specific animal and human sources. In conclusion, B. suis QH05 does not belong to the Brucella epidemic species in China, and as the first isolation of B. suis from yak, this strain expands the host range of B. suis.

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