Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples
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...
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ftdoajarticles:oai:doaj.org/article:1e560d116f5d4f30b8398e2112f7e961 2023-05-15T15:12:32+02:00 Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples Noam B. Teyssier Anna Chen Elias M. Duarte Rene Sit Bryan Greenhouse Sofonias K. Tessema 2021-02-01T00:00:00Z https://doi.org/10.1186/s12936-021-03630-4 https://doaj.org/article/1e560d116f5d4f30b8398e2112f7e961 EN eng BMC https://doi.org/10.1186/s12936-021-03630-4 https://doaj.org/toc/1475-2875 doi:10.1186/s12936-021-03630-4 1475-2875 https://doaj.org/article/1e560d116f5d4f30b8398e2112f7e961 Malaria Journal, Vol 20, Iss 1, Pp 1-8 (2021) Malaria P. falciparum Dried blood spots Tween-Chelex McrBC Selective whole genome amplification Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 article 2021 ftdoajarticles https://doi.org/10.1186/s12936-021-03630-4 2022-12-31T10:20:47Z 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. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Malaria Journal 20 1 |
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ftdoajarticles |
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English |
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Malaria P. falciparum Dried blood spots Tween-Chelex McrBC Selective whole genome amplification Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
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Malaria P. falciparum Dried blood spots Tween-Chelex McrBC Selective whole genome amplification Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 Noam B. Teyssier Anna Chen Elias M. Duarte Rene Sit Bryan Greenhouse Sofonias K. Tessema Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples |
topic_facet |
Malaria P. falciparum Dried blood spots Tween-Chelex McrBC Selective whole genome amplification Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
description |
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. |
format |
Article in Journal/Newspaper |
author |
Noam B. Teyssier Anna Chen Elias M. Duarte Rene Sit Bryan Greenhouse Sofonias K. Tessema |
author_facet |
Noam B. Teyssier Anna Chen Elias M. Duarte Rene Sit Bryan Greenhouse Sofonias K. Tessema |
author_sort |
Noam B. Teyssier |
title |
Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples |
title_short |
Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples |
title_full |
Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples |
title_fullStr |
Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples |
title_full_unstemmed |
Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples |
title_sort |
optimization of whole-genome sequencing of plasmodium falciparum from low-density dried blood spot samples |
publisher |
BMC |
publishDate |
2021 |
url |
https://doi.org/10.1186/s12936-021-03630-4 https://doaj.org/article/1e560d116f5d4f30b8398e2112f7e961 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Malaria Journal, Vol 20, Iss 1, Pp 1-8 (2021) |
op_relation |
https://doi.org/10.1186/s12936-021-03630-4 https://doaj.org/toc/1475-2875 doi:10.1186/s12936-021-03630-4 1475-2875 https://doaj.org/article/1e560d116f5d4f30b8398e2112f7e961 |
op_doi |
https://doi.org/10.1186/s12936-021-03630-4 |
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Malaria Journal |
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20 |
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1 |
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1766343205239914496 |