A new and efficient enrichment method for metagenomic sequencing of Monkeypox virus.
Genome sequencing
Host DNA depletion
Human monkeypox (hMPX)
Metagenomics
Monkeypox virus (MPXV)
Viral surveillance
Journal
BMC genomics
ISSN: 1471-2164
Titre abrégé: BMC Genomics
Pays: England
ID NLM: 100965258
Informations de publication
Date de publication:
17 Jan 2023
17 Jan 2023
Historique:
received:
28
07
2022
accepted:
04
01
2023
entrez:
17
1
2023
pubmed:
18
1
2023
medline:
20
1
2023
Statut:
epublish
Résumé
The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach. A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%. Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.
Sections du résumé
BACKGROUND
BACKGROUND
The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach.
RESULTS
RESULTS
A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%.
CONCLUSIONS
CONCLUSIONS
Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.
Identifiants
pubmed: 36650445
doi: 10.1186/s12864-023-09114-w
pii: 10.1186/s12864-023-09114-w
pmc: PMC9847149
doi:
Substances chimiques
DNA, Viral
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
29Subventions
Organisme : SERGAS-Galician Healthcare Service
ID : Innova Saúde
Organisme : SERGAS-Galician Healthcare Service
ID : Innova Saúde
Organisme : SERGAS-Galician Healthcare Service
ID : Innova Saúde
Organisme : SERGAS-Galician Healthcare Service
ID : Innova Saúde
Organisme : Instituto de Salud Carlos III
ID : CIBERINFEC
Organisme : Instituto de Salud Carlos III
ID : PI20/00413
Organisme : Instituto de Salud Carlos III
ID : CIBERINFEC
Informations de copyright
© 2023. The Author(s).
Références
Genome Biol. 2021 Sep 14;22(1):266
pubmed: 34521459
Sci Rep. 2022 Jun 24;12(1):10768
pubmed: 35750759
Infect Dis Clin North Am. 2019 Dec;33(4):1027-1043
pubmed: 30981594
J Clin Microbiol. 2016 Apr;54(4):919-27
pubmed: 26763966
Nucleic Acids Res. 2021 Jul 2;49(W1):W293-W296
pubmed: 33885785
Bioinformatics. 2016 Oct 1;32(19):3047-8
pubmed: 27312411
Nat Biotechnol. 2019 Jul;37(7):783-792
pubmed: 31235920
Nat Med. 2022 Aug;28(8):1569-1572
pubmed: 35750157
Bioinformatics. 2018 Sep 15;34(18):3094-3100
pubmed: 29750242
Microbiol Resour Announc. 2020 Mar 5;9(10):
pubmed: 32139560
Bioinformatics. 2014 Aug 1;30(15):2114-20
pubmed: 24695404
Bioinformatics. 2014 May 1;30(9):1312-3
pubmed: 24451623
Bioinformatics. 2020 Feb 15;36(4):1303-1304
pubmed: 31553437
PLoS Comput Biol. 2017 Jun 8;13(6):e1005595
pubmed: 28594827
J Gen Microbiol. 1962 Nov;29:523-9
pubmed: 14004140
PLoS One. 2016 Oct 5;11(10):e0163962
pubmed: 27706213
Euro Surveill. 2022 Jul;27(29):
pubmed: 35866435
J Comput Biol. 2012 May;19(5):455-77
pubmed: 22506599
PLoS Comput Biol. 2022 Jan 24;18(1):e1009802
pubmed: 35073327
Bioinformatics. 2009 Aug 15;25(16):2078-9
pubmed: 19505943
Respir Res. 2019 Nov 27;20(1):265
pubmed: 31775777
Bull World Health Organ. 1980;58(2):165-82
pubmed: 6249508
Genome Biol. 2019 Jan 8;20(1):8
pubmed: 30621750
Genome Biol. 2019 Nov 28;20(1):257
pubmed: 31779668
Brief Bioinform. 2008 Jul;9(4):286-98
pubmed: 18372315
Lancet Infect Dis. 2018 Mar;18(3):246
pubmed: 29361427