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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2021-02-01
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| Series: | Malaria Journal |
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| Online Access: | https://doi.org/10.1186/s12936-021-03630-4 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-20T00:02:01Z |
| format | Article |
| id | doaj.art-1e560d116f5d4f30b8398e2112f7e961 |
| institution | Directory Open Access Journal |
| issn | 1475-2875 |
| language | English |
| last_indexed | 2024-12-20T00:02:01Z |
| publishDate | 2021-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Malaria Journal |
| spelling | doaj.art-1e560d116f5d4f30b8398e2112f7e9612022-12-21T20:00:48ZengBMCMalaria Journal1475-28752021-02-012011810.1186/s12936-021-03630-4Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samplesNoam B. Teyssier0Anna Chen1Elias M. Duarte2Rene Sit3Bryan Greenhouse4Sofonias K. Tessema5Department of Medicine, EPPIcenter, University of CaliforniaDepartment of Medicine, EPPIcenter, University of CaliforniaDepartment of Medicine, EPPIcenter, University of CaliforniaChan Zuckerberg BiohubDepartment of Medicine, EPPIcenter, University of CaliforniaDepartment of Medicine, EPPIcenter, University of CaliforniaAbstract 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.https://doi.org/10.1186/s12936-021-03630-4MalariaP. falciparumDried blood spotsTween-ChelexMcrBCSelective whole genome amplification |
| spellingShingle | 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 Malaria Journal Malaria P. falciparum Dried blood spots Tween-Chelex McrBC Selective whole genome amplification |
| title | 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_short | 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 |
| topic | Malaria P. falciparum Dried blood spots Tween-Chelex McrBC Selective whole genome amplification |
| url | https://doi.org/10.1186/s12936-021-03630-4 |
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