Selective whole-genome sequencing ofPlasmodiumparasites directly from blood samples by Nanopore adaptive sampling

Abstract

BackgroundWhole-genome sequencing (WGS) is becoming an increasingly popular tool to study the population genetics and drug resistance ofPlasmodiumspp. However, the predominance of human DNA in a malaria patient blood sample requires time-consuming lab procedures to filter out human DNA or enrichPlasmodiumDNA. Here, we investigated the potential of adaptive sampling to enrich forPlasmodiumDNA while sequencing unenriched patient blood samples on a minION device.ResultsTo compare adaptive sampling versus regular sequencing, a dilution series consisting of 0% up to 100%P. falciparumDNA in human DNA was sequenced. Half of the flowcell channels were run in adaptive sampling mode, enriching for theP. falciparumreference genome, resulting in a 3.2 fold enrichment ofP. falciparumbases on average. Samples with a lower concentration of parasite DNA had a higher enrichment potential. We confirmed these findings by sequencing twoP. falciparumpatient blood samples with common levels of parasitaemia (0.1% and 0.2%). The estimated enrichment was 3.9 and 5.8, which was sufficient to cover at least 97% of theP. falciparumreference genome at a median depth of 20 (highest parasitaemia) or 5 (lowest parasitaemia). A comparison of 38 drug resistance variants (WHO) obtained via adaptive sequencing or Sanger sequencing showed a high concordance between the two methods, suggesting that the obtained sequencing data is of sufficient quality to address common clinical research questions for patients with parasitaemias of 0.1% and higher.ConclusionsOur results demonstrate that adaptive Nanopore sequencing has the potential to replace more time-consumingPlasmodium-enrichment protocols and sequence directly from patient blood, given further improvements in cost-efficiency.

Nanopore adaptive sampling to identify the NLR-gene family in melon (Cucumis meloL.)

“…We obtained an enrichment by yield up to 3.7 times on average and an enrichment by selection up to 69 times on average, even when the reference was genetically distant from the sequenced accession. These findings are comparable to the best results previously obtained enriching individuals in metagenomics samples (De Meulenaere et al, 2022;Martin et al, 2022) and outperformed the enrichment values previously obtained with loci panels (Hogers et al, 2020). The successful enrichment with NAS could be linked to the percentage of the genome targeted here (~1.41%), the size of the targets, or the size of the DNA fragments, as they have been demonstrated to be main factors of enrichment rate (Martin et al, 2022;Community Nanopore, 2023).…”

“…One key factor that largely affects final yield in ONT sequencing runs is the number of active channels at the beginning of the run and their lifespan, which may significantly differ from one flowcell to another. Therefore, we implemented a half-flowcell design to compare NAS and WGS eliminating biases that may arise when using two different flowcells, as previously settled in many studies (De Meulenaere et al, 2022; Martin et al, 2022). Results showed that for both Anso77 and Doublon, NAS produced about four times more on-target data than WGS (Table 3), while generating about ten times less total data (see filtered NAS and WGS in Table 2).…”

“…NAS requires a standard library preparation, eliminates the need for DNA amplification, circumvents laborious or expensive experimental design or probes synthesis, and offers real-time selective enrichment (Martin et al, 2022; Miyatake et al, 2022). NAS has been used in clinical settings and for the enrichment of metagenomic samples (Kipp et al, 2021; De Meulenaere et al, 2022; Martin et al, 2022; Greer et al, 2023; Hewel et al, 2023; Su et al, 2023; Wrenn & Drown, 2023). Therefore, NAS emerges as a promising approach for studying target regions, especially those that are highly complex, such as disease-associated repeat loci in humans (Miyatake et al, 2022; Stevanovski et al, 2022).…”

A newPlasmodium vivaxreference genome for South American isolates

“…However, the here used combination of long-read sequencing and a low-input material approach, might facilitate assembly of P. vivax field isolates from blood volumes as small as a finger prick. In addition, Oxford Nanopore’s portable sequencer could become a quick and easy to use alternative for PacBio sequencing (De Meulenaere et al, 2022a), given further improvements in accuracy and DNA input requirements.…”

Flexible and cost-effective genomic surveillance ofP. falciparummalaria with targeted nanopore sequencing

“…1-2kbp). Moreover, work on adaptive sampling of reads during nanopore sequencing has recently been applied to P. falciparum malaria 61 , and variations of this approach may help recover better data from low parasitemia samples.…”