scholarly journals Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

2021 ◽  
Author(s):  
Noam Teyssier ◽  
Anna Chen ◽  
Elias Duarte ◽  
Rene Sit ◽  
Bryan Greenhouse ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Dropout Rate ◽  
Dried Blood Spots ◽  
High Ratio ◽  
Low Density ◽  
Whole Genome ◽  
Cross Sectional ◽  
Modest Improvement ◽  
Primer Sets

Abstract Background Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. The effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification were evaluated on the quality and fidelity of WGS data recovered from DBS.MethodsLow parasite density mock DBS samples were created, extracted either with Tween-Chelex or QIAamp, treated with or without McrBC, and amplified with one of three different amplification techniques (two sWGA primer sets and one rWGA). Extraction conditions were evaluated on performance of sequencing depth, percentiles of coverage, and expected SNP concordance.ResultsAt 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5X depth = 93% genome) than QIAamp extracted samples (5X depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published.ConclusionsOverall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

scholarly journals Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Noam B. Teyssier ◽  
Anna Chen ◽  
Elias M. Duarte ◽  
Rene Sit ◽  
Bryan Greenhouse ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Dropout Rate ◽  
Dried Blood Spots ◽  
High Ratio ◽  
Low Density ◽  
Whole Genome ◽  
Cross Sectional ◽  
Modest Improvement ◽  
Primer Sets

Abstract Background Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. The effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification were evaluated on the quality and fidelity of WGS data recovered from DBS. Methods Low parasite density mock DBS samples were created, extracted either with Tween-Chelex or QIAamp, treated with or without McrBC, and amplified with one of three different amplification techniques (two sWGA primer sets and one rWGA). Extraction conditions were evaluated on performance of sequencing depth, percentiles of coverage, and expected SNP concordance. Results At 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5 × depth = 93% genome) than QIAamp extracted samples (5 × depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. Conclusions Overall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

scholarly journals Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

2021 ◽  
Author(s):  
Noam Teyssier ◽  
Anna Chen ◽  
Elias Duarte ◽  
Rene Sit ◽  
Bryan Greenhouse ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Dropout Rate ◽  
Dried Blood Spots ◽  
High Ratio ◽  
Low Density ◽  
Whole Genome ◽  
Cross Sectional ◽  
Modest Improvement ◽  
Primer Sets

Abstract Background: Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. We evaluated the effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification on the quality and fidelity of WGS data recovered from DBS.Results: At 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5X depth = 93% genome) than QIAamp extracted samples (5X depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. Conclusions: Overall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

scholarly journals Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

2019 ◽  
Author(s):  
Noam B. Teyssier ◽  
Anna Chen ◽  
Elias M Duarte ◽  
Rene Sit ◽  
Bryan Greenhouse ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Dropout Rate ◽  
Dried Blood Spots ◽  
High Ratio ◽  
Low Density ◽  
Whole Genome ◽  
Cross Sectional ◽  
Modest Improvement ◽  
Primer Sets

AbstractBackgroundWhole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. We evaluated the effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification on the quality and fidelity of WGS data recovered from DBS.ResultsAt 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5X depth = 93% genome) than QIAamp extracted samples (5X depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published.ConclusionsOverall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

scholarly journals Optimization of Whole-Genome Sequencing of Plasmodium Falciparum From Low-Density Dried Blood Spot Samples

2020 ◽  
Author(s):  
Noam Teyssier ◽  
Anna Chen ◽  
Elias Duarte ◽  
Rene Sit ◽  
Bryan Greenhouse ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Dropout Rate ◽  
Dried Blood Spots ◽  
High Ratio ◽  
Low Density ◽  
Whole Genome ◽  
Cross Sectional ◽  
Modest Improvement ◽  
Primer Sets

Abstract Background: Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. We evaluated the effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification on the quality and fidelity of WGS data recovered from DBS. Results: At 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5X depth = 93% genome) than QIAamp extracted samples (5X depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. Conclusions: Overall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

scholarly journals Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

2020 ◽  
Author(s):  
Noam Teyssier ◽  
Anna Chen ◽  
Elias Duarte ◽  
Rene Sit ◽  
Bryan Greenhouse ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Dropout Rate ◽  
Dried Blood Spots ◽  
Low Density ◽  
Whole Genome ◽  
Cross Sectional ◽  
Modest Improvement ◽  
Blood Spots ◽  
Gates Foundation

Abstract Background: Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. We evaluated the effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification on the quality and fidelity of WGS data recovered from DBS. Results: At 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5X depth = 93% genome) than QIAamp extracted samples (5X depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. Conclusions: Overall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys. Keywords: Malaria, P. falciparum, dried blood spots, Tween-Chelex, McrBC, selective whole genome amplification, whole genome sequencing This work was supported by the Bill & Melinda Gates Foundation, Grant Number OPP1132226 This work was supported by the Bill & Melinda Gates Foundation, Grant Number OPP1132226

scholarly journals Somatic Structural Alterations in Childhood Leukemia Can Be Backtracked in Neonatal Dried Blood Spots by Use of Whole-Genome Sequencing and Digital PCR

2019 ◽  
Vol 65 (2) ◽  
pp. 345-347
Author(s):  
Fulya Taylan ◽  
Benedicte Bang ◽  
Ingegerd Ivanov Öfverholm ◽  
Anh-Nhi Tran ◽  
Mats Heyman ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Childhood Leukemia ◽  
Digital Pcr ◽  
Dried Blood Spots ◽  
Whole Genome ◽  
Structural Alterations ◽  
Blood Spots

scholarly journals Digital-WGS: Automated, highly efficient whole-genome sequencing of single cells by digital microfluidics

2020 ◽  
Vol 6 (50) ◽  
pp. eabd6454
Author(s):  
Qingyu Ruan ◽  
Weidong Ruan ◽  
Xiaoye Lin ◽  
Yang Wang ◽  
Fenxiang Zou ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Whole Genome Sequencing ◽  
Single Cell ◽  
Genome Sequencing ◽  
Single Cells ◽  
Digital Microfluidics ◽  
Dropout Rate ◽  
Great Promise ◽  
Whole Genome ◽  
Exogenous Dna

Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.

scholarly journals Causes of false-negative rapid diagnostic tests for symptomatic malaria in the DRC

2020 ◽  
Author(s):  
Jonathan B Parr ◽  
Eddy Kieto ◽  
Fernandine Phanzu ◽  
Paul Masiangi ◽  
Kashamuka Mwandagalirwa ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Falciparum Malaria ◽  
Genome Sequencing ◽  
Diagnostic Tests ◽  
False Negative ◽  
Rapid Diagnostic Tests ◽  
Whole Genome ◽  
Cross Sectional ◽  
Symptomatic Malaria ◽  
Asymptomatic Children

Background The majority of Plasmodium falciparum malaria diagnoses in Africa are made using rapid diagnostic tests (RDTs) that detect histidine-rich protein 2. Increasing reports of false-negative RDT results due to parasites with deletions of the pfhrp2 and/or pfhrp3 genes (pfhrp2/3) raise concern about existing malaria diagnostic strategies. We previously identified pfhrp2-negative parasites among asymptomatic children in the Democratic Republic of the Congo (DRC), but their impact on diagnosis of symptomatic malaria is unknown. Methods We performed a cross-sectional study of false-negative RDTs in symptomatic subjects in 2017. Parasites were characterized by microscopy; RDT; pfhrp2/3 genotyping and species-specific PCR assays; a multiplex bead-based immunoassay; and/or whole-genome sequencing. Results Among 3,627 symptomatic subjects, we identified 427 (11.8%) RDT-/microscopy+ cases. Parasites from eight (0.2%) samples were initially classified as putative pfhrp2/3 deletions by PCR, but antigen testing and whole-genome sequencing confirmed the presence of intact genes. Malaria prevalence was high (57%) and non-falciparum co-infection common (15%). HRP2-based RDT performance was satisfactory and superior to microscopy. Conclusions Symptomatic malaria due to pfhrp2/3-deleted P. falciparum was not observed in the DRC. Ongoing HRP2-based RDT use is appropriate for the detection of falciparum malaria in the DRC.

scholarly journals Rapid and Inexpensive Whole-Genome Sequencing of SARS-CoV-2 using 1200 bp Tiled Amplicons and Oxford Nanopore Rapid Barcoding

2020 ◽  
Author(s):  
Nikki E. Freed ◽  
Markéta Vlková ◽  
Muhammad B. Faisal ◽  
Olin K. Silander
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Variant Calling ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Cost Efficient ◽  
Primer Sets ◽  
Library Preparation Method

AbstractRapid and cost-efficient whole-genome sequencing of SARS-CoV-2, the virus that causes COVID-19, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10,000 reads (approximately 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

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