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 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

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

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 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 Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification

2016 ◽  
Vol 15 (1) ◽  
Author(s):  
Samuel O. Oyola ◽  
Cristina V. Ariani ◽  
William L. Hamilton ◽  
Mihir Kekre ◽  
Lucas N. Amenga-Etego ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome Amplification ◽  
Dried Blood Spots ◽  
Whole Genome ◽  
Genome Amplification ◽  
Blood Spots

scholarly journals Validation of whole genome sequencing from dried blood spots

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Pooja Agrawal ◽  
Shanmukh Katragadda ◽  
Arun K. Hariharan ◽  
Vijayashree Gauribidanur Raghavendrachar ◽  
Arunika Agarwal ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Copy Number ◽  
Large Scale ◽  
Venous Blood ◽  
Dried Blood Spots ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Blood Spots ◽  
Mitochondrial Copy Number

Abstract Background Dried blood spots (DBS) are a relatively inexpensive source of nucleic acids and are easy to collect, transport, and store in large-scale field surveys, especially in resource-limited settings. However, their performance in whole-genome sequencing (WGS) relative to that of venous blood DNA has not been analyzed for various downstream applications. Methods This study compares the WGS performance of DBS paired with venous blood samples collected from 12 subjects. Results Results of standard quality checks of coverage, base quality, and mapping quality were found to be near identical between DBS and venous blood. Concordance for single-nucleotide variants, insertions and deletions, and copy number variants was high between these two sample types. Additionally, downstream analyses typical of population-based studies were performed, such as mitochondrial heteroplasmy detection, haplotype analysis, mitochondrial copy number changes, and determination of telomere lengths. The absolute mitochondrial copy number values were higher for DBS than for venous blood, though the trend in sample-to-sample variation was similar between DBS and blood. Telomere length estimates in most DBS samples were on par with those from venous blood. Conclusion DBS samples can serve as a robust and feasible alternative to venous blood for studies requiring WGS analysis.

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.

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