ASSESSMENT OF BIOSKRYB WHOLE GENOME AMPLIFICATION METHODOLOGY FOR GENOME-WIDE ASSESSMENT FOR SNPs AND INDELS USING ILLUMINA WHOLE GENOME SEQUENCING

AbstractThe MinION was used to evaluate upper respiratory disease infections using both whole genome amplification (WGA), targeted sequencing, and was found to have tremendous potential for field use. The MinION nanopore sequencer was been released to community testers for evaluation using a variety of sequencing applications. The MinION was used to evaluate upper respiratory disease infections using both whole genome amplification and targeted sequencing, and was found to have tremendous potential for field use. In this study, we tested the ability of the MinION nanopore sequencer to accurately identify and differentiate clinical bacterial and viral samples via targeted sequencing and whole genome sequencing. The current nanopore technology has limitations with respect to error rate but has steadily improved with development of new flow cells and kits. Upper respiratory disease organisms were successfully identified and differentiated down to the strain level with 87-98% alignment to our reference genome database. The ability to differentiate strains by amplicon and whole genome sequencing on the MinION was accomplished despite the observed average per 100-base error rate averaged 1.2E-01. This study offers evidence of the utility of sequencing to identify and differentiate both viral and bacterial species present within clinical samples.

Download Full-text

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

mBio ◽

10.1128/mbio.02257-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 28

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.

Download Full-text

Refining the evolutionary time machine: an assessment of whole genome amplification using single historical Daphnia eggs

10.1101/2021.04.19.440325 ◽

2021 ◽

Author(s):

Christopher James O'Grady ◽

Vignesh Dhandapani ◽

John K. Colbourne ◽

Dagmar Frisch

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome Amplification ◽

Natural Populations ◽

Population Level ◽

Freshwater Ecosystems ◽

Whole Genome ◽

Exogenous Dna ◽

Genome Amplification ◽

Dormant Eggs

Whole genome sequencing is instrumental for the study of genome variation in natural populations, delivering important knowledge on genomic modifications and potential targets of natural selection at the population level. Large dormant eggbanks of aquatic invertebrates such as the keystone herbivore Daphnia, a microcrustacean widespread in freshwater ecosystems, provide detailed sedimentary archives to study genomic processes over centuries. To overcome the problem of limited DNA amounts in single Daphnia dormant eggs, we developed an optimised workflow for whole genome amplification (WGA), yielding sufficient amounts of DNA for downstream whole genome sequencing of individual historical eggs, including polyploid lineages. We compare two WGA kits, applied to recently produced Daphnia magna dormant eggs from laboratory cultures, and to historical dormant eggs of Daphnia pulicaria collected from Arctic lake sediment between 10y and 300y old. Resulting genome coverage breadth in most samples was ~70%, including those from >100y old isolates. Sequence read distribution was highly correlated among samples amplified with the same kit, but less correlated between kits. Despite this, a high percentage of genomic positions with SNPs in one or more samples (maximum of 74% between kits, and 97% within kits) were recovered at a depth required for genotyping. As a by-product of sequencing we obtained 100% coverage of the mitochondrial genomes even from the oldest isolates (~300y). The mtDNA provides an additional source for evolutionary studies of these populations. We provide an optimised workflow for WGA followed by whole genome sequencing including steps to minimise exogenous DNA.

Download Full-text

Selective whole genome amplification as a tool to enrich specimens with low Treponema pallidum genomic DNA copies for whole genome sequencing

10.1101/2021.07.09.451864 ◽

2021 ◽

Author(s):

Charles Michael Thurlow ◽

Sandeep J Joseph ◽

Lilia M Ganova-Raeva ◽

Samantha Katz ◽

Lara Pereira ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome Amplification ◽

Multiple Displacement Amplification ◽

Treponema Pallidum ◽

The United States ◽

Whole Genome ◽

Genome Amplification ◽

Clinical Specimens ◽

Enrichment Methods

Downstream next generation sequencing of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing Multiple Displacement Amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA followed by MDA as enrichment methods to improve the yields of T. pallidum DNA in rabbit propagated isolates and lesion specimens from patients with primary and secondary syphilis. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/μl input for SWGA and >129 genomic copies/μl for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now.

Download Full-text