Comparison of sequencing analyses targeting the V1/V2 and V3/V4
regions in the 16S rDNA gene – analysis of bacteriome in oral
swabs and esophageal tissues

a 2023

Comparison of sequencing analyses targeting the V1/V2 and V3/V4 regions in the 16S rDNA gene – analysis of bacteriome in oral swabs and esophageal tissues

MLČŮCHOVÁ, Natálie, Terezie SLÁMOVÁ, Petra BRENEROVÁ, Tereza DEISSOVÁ, Jan BÖHM et. al.

Basic information

Original name

Comparison of sequencing analyses targeting the V1/V2 and V3/V4 regions in the 16S rDNA gene – analysis of bacteriome in oral swabs and esophageal tissues

Authors

MLČŮCHOVÁ, Natálie (203 Czech Republic), Terezie SLÁMOVÁ (203 Czech Republic), Petra BRENEROVÁ (203 Czech Republic), Tereza DEISSOVÁ (203 Czech Republic), Jan BÖHM (203 Czech Republic), Lumír KUNOVSKÝ (203 Czech Republic), Radek KROUPA (203 Czech Republic), Jiří DOLINA (203 Czech Republic), Vladimír PROCHÁZKA (203 Czech Republic), Tomáš GROLICH (203 Czech Republic), Jitka VACULOVÁ (203 Czech Republic), Petr ANDRLA (203 Czech Republic), Vít NAVRÁTIL, Ondřej URBAN (203 Czech Republic), Tomáš HARUŠTIAK, Robert LISCHKE (203 Czech Republic), Lydie IZAKOVIČOVÁ HOLLÁ (203 Czech Republic), Ondřej SLABÝ (203 Czech Republic), Zdeněk KALA (203 Czech Republic), Eva BUDINSKÁ (703 Slovakia) and Petra BOŘILOVÁ LINHARTOVÁ (203 Czech Republic, guarantor, belonging to the institution)

Edition

Biologické dny, 2023, 2023

Other information

Language

English

Type of outcome

Konferenční abstrakt

Field of Study

10608 Biochemistry and molecular biology

Country of publisher

Czech Republic

Confidentiality degree

není předmětem státního či obchodního tajemství

RIV identification code

RIV/00216224:14310/23:00133534

Organization unit

Faculty of Science

Keywords in English

V1V2 region; V3V4 region; 16S rRNA; next-generation sequencing; microbiome analysis; gastrointestinal tract

Změněno: 15/2/2024 09:34, Mgr. Terezie Slámová

Abstract

V originále

Objective: Bacterial 16S ribosomal DNA (rDNA) sequences are widely used to determine taxonomic and phylogenetic classification of human bacteriome. The selection of the hypervariable sub-regions to be analyzed may affect the analysis results, which may lead to incorrect interpretation of data. Selection of appropriate validated methodology in next-generation sequencing using 16S rRNA is, therefore, crucial. This work aimed to compare results obtained from 16S rRNA sequencing analysis of V1/V2 vs. V3/V4 region. Methods: Six patients with BE/EAC were included in this study. DNA from six oral swabs and twelve esophageal biopsies from Barrett's mucosa/adenocarcinoma and from healthy sites were isolated using the AllPrep DNA/RNA Kit (Qiagen). The V1/V2 region of the 16S rRNA gene was amplified using PCR and modified primers 68Fmod and 338R. The metagenomic library was prepared using the MiniSeq® High Output Kit (2 x 150 paired end sequencing) and deeply sequenced on an Illumina Miniseq 150bp instrument.    The V3/V4 16S rRNA sequencing analysis was performed on the same samples, which were, in addition, spiked by a mock community (Allobacillus and Imtechella, microbial concentration 104; ZymoBIOMICS Spike-in Control I, High Microbial Load); six negative controls (NC, DNA-free water) were added to the analysis. The V3/V4 region of the gene for 16S rRNA was amplified using Illumina sequencing primers. The metagenomic library was prepared using the Nextera DNA Flex Library Prep Kit V3 and deeply sequenced on an Illumina MiSeq instrument.   Results: The number of reads per sample was generally higher in samples sequenced using the approach focusing on V3/V4, which led to the higher resolving power than the V1/V2 method. In other words, more taxa were identified by V3/V4 16S rRNA sequencing than by V1/V2 16S rRNA sequencing. The MDS Canberra distance analysis showed a regular shift between V1/V2 and V3/V4 analyses, which indicates that both methods yield a similar trend with a systematic difference rather than random error. Conclusion: In studied matrices, the relative abundance of taxa using 16S rRNA sequencing of the V3/V4 region were more consistent with literature (i.e., detected a high abundance of Streptococcus) than the V1/V2 method. We, therefore, conclude that the V3/V4 approach with internal standard is more suitable for analysis of oral and esophageal bacteriome than the V1/V2 method.

Links

LM2018121, research and development project

Name: Výzkumná infrastruktura RECETOX (Acronym: RECETOX RI)

Investor: Ministry of Education, Youth and Sports of the CR, RECETOX RI

NU20-03-00126, research and development project

Name: Hostitelský mikrobiom ve vztahu k rozvoji Barrettova jícnu a adenokarcinomu jícnu

Investor: Ministry of Health of the CR, Host microbiome in relation to Barrett ́s esophagus and esophageal adenocarcinoma development, Subprogram 1 - standard

Citovat

MLČŮCHOVÁ, Natálie, Terezie SLÁMOVÁ, Petra BRENEROVÁ, Tereza DEISSOVÁ, Jan BÖHM, Lumír KUNOVSKÝ, Radek KROUPA, Jiří DOLINA, Vladimír PROCHÁZKA, Tomáš GROLICH, Jitka VACULOVÁ, Petr ANDRLA, Vít NAVRÁTIL, Ondřej URBAN, Tomáš HARUŠTIAK, Robert LISCHKE, Lydie IZAKOVIČOVÁ HOLLÁ, Ondřej SLABÝ, Zdeněk KALA, Eva BUDINSKÁ and Petra BOŘILOVÁ LINHARTOVÁ. Comparison of sequencing analyses targeting the V1/V2 and V3/V4 regions in the 16S rDNA gene – analysis of bacteriome in oral swabs and esophageal tissues. In Biologické dny, 2023. 2023.

@proceedings{2374078,
   author = {Mlčůchová, Natálie and Slámová, Terezie and Brenerová, Petra and Deissová, Tereza and Böhm, Jan and Kunovský, Lumír and Kroupa, Radek and Dolina, Jiří and Procházka, Vladimír and Grolich, Tomáš and Vaculová, Jitka and Andrla, Petr and Navrátil, Vít and Urban, Ondřej and Haruštiak, Tomáš and Lischke, Robert and Izakovičová Hollá, Lydie and Slabý, Ondřej and Kala, Zdeněk and Budinská, Eva and Bořilová Linhartová, Petra},
   booktitle = {Biologické dny, 2023},
   keywords = {V1V2 region; V3V4 region; 16S rRNA; next-generation sequencing; microbiome analysis; gastrointestinal tract},
   language = {eng},
   title = {Comparison of sequencing analyses targeting the V1/V2 and V3/V4 regions in the 16S rDNA gene – analysis of bacteriome in oral swabs and esophageal tissues},
   year = {2023}
}

TY  - CONF
ID  - 2374078
AU  - Mlčůchová, Natálie - Slámová, Terezie - Brenerová, Petra - Deissová, Tereza - Böhm, Jan - Kunovský, Lumír - Kroupa, Radek - Dolina, Jiří - Procházka, Vladimír - Grolich, Tomáš - Vaculová, Jitka - Andrla, Petr - Navrátil, Vít - Urban, Ondřej - Haruštiak, Tomáš - Lischke, Robert - Izakovičová Hollá, Lydie - Slabý, Ondřej - Kala, Zdeněk - Budinská, Eva - Bořilová Linhartová, Petra
PY  - 2023
TI  - Comparison of sequencing analyses targeting the V1/V2 and V3/V4 regions in the 16S rDNA gene – analysis of bacteriome in oral swabs and esophageal tissues
KW  - V1V2 region
KW  - V3V4 region
KW  - 16S rRNA
KW  - next-generation sequencing
KW  - microbiome analysis
KW  - gastrointestinal tract
N2  - Objective: Bacterial 16S ribosomal DNA (rDNA) sequences are widely used to determine taxonomic and phylogenetic classification of human bacteriome. The selection of the hypervariable sub-regions to be analyzed may affect the analysis results, which may lead to incorrect interpretation of data. Selection of appropriate validated methodology in next-generation sequencing using 16S rRNA is, therefore, crucial. This work aimed to compare results obtained from 16S rRNA sequencing analysis of V1/V2 vs. V3/V4 region. Methods: Six patients with BE/EAC were included in this study. DNA from six oral swabs and twelve esophageal biopsies from Barrett's mucosa/adenocarcinoma and from healthy sites were isolated using the AllPrep DNA/RNA Kit (Qiagen). The V1/V2 region of the 16S rRNA gene was amplified using PCR and modified primers 68Fmod and 338R. The metagenomic library was prepared using the MiniSeq® High Output Kit (2 x 150 paired end sequencing) and deeply sequenced on an Illumina Miniseq 150bp instrument.    The V3/V4 16S rRNA sequencing analysis was performed on the same samples, which were, in addition, spiked by a mock community (Allobacillus and Imtechella, microbial concentration 104; ZymoBIOMICS Spike-in Control I, High Microbial Load); six negative controls (NC, DNA-free water) were added to the analysis. The V3/V4 region of the gene for 16S rRNA was amplified using Illumina sequencing primers. The metagenomic library was prepared using the Nextera DNA Flex Library Prep Kit V3 and deeply sequenced on an Illumina MiSeq instrument.   Results: The number of reads per sample was generally higher in samples sequenced using the approach focusing on V3/V4, which led to the higher resolving power than the V1/V2 method. In other words, more taxa were identified by V3/V4 16S rRNA sequencing than by V1/V2 16S rRNA sequencing. The MDS Canberra distance analysis showed a regular shift between V1/V2 and V3/V4 analyses, which indicates that both methods yield a similar trend with a systematic difference rather than random error. Conclusion: In studied matrices, the relative abundance of taxa using 16S rRNA sequencing of the V3/V4 region were more consistent with literature (i.e., detected a high abundance of Streptococcus) than the V1/V2 method. We, therefore, conclude that the V3/V4 approach with internal standard is more suitable for analysis of oral and esophageal bacteriome than the V1/V2 method.
ER  -

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