16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14110%2F23%3A00132245" target="_blank" >RIV/00216224:14110/23:00132245 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.nature.com/articles/s41598-023-39575-8" target="_blank" >https://www.nature.com/articles/s41598-023-39575-8</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1038/s41598-023-39575-8" target="_blank" >10.1038/s41598-023-39575-8</a>

Jazyk výsledku

angličtina

Název v původním jazyce

16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling

Popis výsledku v původním jazyce

16S rRNA amplicon sequencing or, more recently, metatranscriptomic analysis are currently the only preferred methods for microbial profiling of samples containing a predominant ratio of human to bacterial DNA. However, due to the off-target amplification of human DNA, current protocols are inadequate for bioptic samples. Here we present an efficient, reliable, and affordable method for the bacteriome analysis of clinical samples human DNA content predominates. We determined the microbiota profile in a total of 40 human biopsies of the esophagus, stomach, and duodenum using 16S rRNA amplicon sequencing with the widely used 515F-806R (V4) primers targeting the V4 region, 68F-338R primers and a modified set of 68F-338R (V1-V2M) primers targeting the V1-V2 region. With the V4 primers, on average 70% of amplicon sequence variants (ASV) mapped to the human genome. On the other hand, this off-target amplification was absent when using the V1-V2M primers. Moreover, the V1-V2M primers provided significantly higher taxonomic richness and reproducibility of analysis compared to the V4 primers. We conclude that the V1-V2M 16S rRNA sequencing method is reliable, cost-effective, and applicable for low-bacterial abundant human samples in medical research.

Název v anglickém jazyce

16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling

Popis výsledku anglicky

16S rRNA amplicon sequencing or, more recently, metatranscriptomic analysis are currently the only preferred methods for microbial profiling of samples containing a predominant ratio of human to bacterial DNA. However, due to the off-target amplification of human DNA, current protocols are inadequate for bioptic samples. Here we present an efficient, reliable, and affordable method for the bacteriome analysis of clinical samples human DNA content predominates. We determined the microbiota profile in a total of 40 human biopsies of the esophagus, stomach, and duodenum using 16S rRNA amplicon sequencing with the widely used 515F-806R (V4) primers targeting the V4 region, 68F-338R primers and a modified set of 68F-338R (V1-V2M) primers targeting the V1-V2 region. With the V4 primers, on average 70% of amplicon sequence variants (ASV) mapped to the human genome. On the other hand, this off-target amplification was absent when using the V1-V2M primers. Moreover, the V1-V2M primers provided significantly higher taxonomic richness and reproducibility of analysis compared to the V4 primers. We conclude that the V1-V2M 16S rRNA sequencing method is reliable, cost-effective, and applicable for low-bacterial abundant human samples in medical research.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10700 - Other natural sciences

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Nature Scientific Reports

ISSN

2045-2322

e-ISSN

—

Svazek periodika

13

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

10

Strana od-do

1-10

Kód UT WoS článku

001042854100051

EID výsledku v databázi Scopus

2-s2.0-85166745396