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New and environment friendly technique Monkeypox virus sequencing

New and environment friendly technique Monkeypox virus sequencing
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In a current research posted to the medRxiv* preprint server, Spanish researchers described a brand new enrichment technique for metagenomic sequencing of the monkeypox virus (MPXV).

Human monkeypox is a zoonotic illness that originated within the central and west African jungles and was first reported within the Democratic Republic of Congo in 1970. Monkeys and rodents unfold the illness, though the unique reservoir stays unknown. People contract MPXV an infection by way of contact with contaminated animals/people and contaminated supplies. MPXV is an enveloped virus with roughly 197 kb of double-stranded (ds) DNA.

MPXV belongs to the Orthopoxvirus genus underneath the Poxviridae household of double-stranded DNA viruses. Genome sequencing has recognized two clades of MPXV: Congo Basin (CB) and West African (WA) clades. The CB clade causes extreme illness and better mortality. Typically, poxviruses (together with MPXV) exhibit better tolerance to a variety of pH and are rather more proof against desiccation than different enveloped viruses. In addition to, they’re much less delicate to disinfectants than different enveloped viruses.

New and environment friendly technique Monkeypox virus sequencingExamine: A brand new and environment friendly enrichment technique for metagenomic sequencing of monkeypox virus Picture Credit score: NIAID

Concerning the research

Within the current research, researchers evaluated a brand new technique of MPXV DNA enrichment compared to the non-enrichment method. Medical specimens from lesions or vesicular fluid swabs have been obtained, and two MPXV-positive samples (MP01 and MP03) have been chosen. DNA extraction was carried out by two totally different protocols.

Within the first (non-enrichment) technique, DNA was extracted utilizing MagNA pure compact nucleic acid isolation equipment I. The second protocol was modified from a saponin-based differential lysis approach and centrifuged at excessive g-force (35,000 g). This technique was designed to complement MPXV DNA samples to keep away from losing sequencing quota.

A tender centrifugation step (at low g-force) was carried out initially to take away giant particles. Host DNA was depleted with the remedy of saponin, sodium chloride (NaCl), and deoxyribonuclease (DNase). NaCl, DNase, and saponin have been eliminated for sequencing. The samples have been processed utilizing the non-enrichment (MP01bCHUAC, MP03bCHUAC) and enrichment strategies (MP01CHUAC, MP03CHUAC).

Phylogenomic tree.  Phylogenomic analysis of the present study's samples, comparing them to all complete MPXV genomes available in GenBank to date (2022-07-18, 275 genomes from taxid 10244).  This does not pretend to infer the evolution of the virus, only to locate the most similar entries to the samples in this study.  A color strip indicates each sample's lineage (A: purple, A.1.1: yellow, A.2: green, B.1: blue), and orange areas highlight the study's samples.Phylogenomic tree. Phylogenomic evaluation of the current research’s samples, evaluating them to all full MPXV genomes out there in GenBank to this point (2022-07-18, 275 genomes from taxid 10244). This doesn’t faux to deduce the evolution of the virus, solely to find probably the most related entries to the samples on this research. A coloration strip signifies every pattern’s lineage (A: purple, A.1.1: yellow, A.2: inexperienced, B.1: blue), and orange areas spotlight the research’s samples.

Findings

Within the preliminary outcomes by way of the Kraken 2 taxonomic classification system utilizing uncooked unfiltered reads, the authors famous that almost all reads within the enriched samples (MP01CHUAC and MP03CHUAC) belonged to MPXV, and nearly none have been categorized as host contamination. In distinction, most reads in non-enriched samples (MP01bCHUAC and MP03bCHUAC) have been of human DNA.

When Finest Match Tagger (BMTagger) eliminated human DNA contamination, the learn counts for MP01CHUAC and MP03CHUAC lowered by 1% to 2 %, whereas the discount for MP01bCHUAC and MP03bCHUAC was 90% to 95%. Within the subsequent step of high quality management, any of the reads not categorized as Orthopoxvirus was eliminated. This resulted in much less drastic change and skim counts decreased by 30% for enriched samples and eight% – 41% for non-enriched samples.

In comparison with uncooked/unique reads, learn counts after the ultimate high quality management step decreased by 50% for enriched samples and 93% – 98% for non-enriched samples. The remaining reads have been aligned to a reference sequence the place reads from enriched samples had a median depth of 1500 – 1800, whereas these from non-enriched samples had a median depth of 80 – 100.

However, all samples produced a high quality consensus sequence of the MPXV B.1 lineage. MP01CHUAC had one nucleotide mutation, whereas MP03CHUAC had two amino acid substitutions in opposition to the reference genome.

Conclusions

The research discovered important variations between each protocols when evaluating depth and skim counts. The lower in learn counts when host reads have been eliminated was large (90% to 95%) for non-enrichment protocol however marginal (1% to 2%) for enrichment protocol. As well as, the enrichment technique retained half the reads after the ultimate high quality management step, whereas the non-enrichment protocol saved solely 2% – 3% of the unique reads.

After aligning the cleaned reads to a reference MPXV sequence, the median depth was greater for enriched samples than for non-enriched samples. All samples generated good high quality consensus primarily based on alignment, however the elevated depth (with enrichment protocol) is important to belief the noticed genetic adjustments.

In conclusion, the brand new enrichment technique considerably improved sequencing effectivity, variety of viral reads, depth, and the trustworthiness of consensus sequences. Notably, eradicating host sequences earlier than sequencing permits for the inclusion of extra samples per cartridge, decreasing prices and time for analysis and rising the effectivity of sequencing.

*Vital discover

medRxiv publishes preliminary scientific reviews that aren’t peer-reviewed and, subsequently, shouldn’t be thought to be conclusive, information scientific follow/health-related conduct, or handled as established data.

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