16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling
Abstract 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...
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| Format: | Article |
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Nature Portfolio
2023-08-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-39575-8 |
| _version_ | 1797453002722770944 |
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| author | Tereza Deissová Martina Zapletalová Lumír Kunovský Radek Kroupa Tomáš Grolich Zdeněk Kala Petra Bořilová Linhartová Jan Lochman |
| author_facet | Tereza Deissová Martina Zapletalová Lumír Kunovský Radek Kroupa Tomáš Grolich Zdeněk Kala Petra Bořilová Linhartová Jan Lochman |
| author_sort | Tereza Deissová |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-09T15:16:35Z |
| format | Article |
| id | doaj.art-6e88d9de7db1466abdf282c7232a13ce |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-09T15:16:35Z |
| publishDate | 2023-08-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj.art-6e88d9de7db1466abdf282c7232a13ce2023-11-26T13:03:14ZengNature PortfolioScientific Reports2045-23222023-08-0113111010.1038/s41598-023-39575-816S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profilingTereza Deissová0Martina Zapletalová1Lumír Kunovský2Radek Kroupa3Tomáš Grolich4Zdeněk Kala5Petra Bořilová Linhartová6Jan Lochman7Department of Biochemistry, Faculty of Science, Masaryk UniversityDepartment of Biochemistry, Faculty of Science, Masaryk UniversityDepartment of Gastroenterology and Internal Medicine, University Hospital Brno, and Faculty of Medicine, Masaryk, UniversityDepartment of Gastroenterology and Internal Medicine, University Hospital Brno, and Faculty of Medicine, Masaryk, UniversityDepartment of Gastroenterology and Internal Medicine, University Hospital Brno, and Faculty of Medicine, Masaryk, UniversityDepartment of Surgery, University Hospital Brno, and Faculty of Medicine, Masaryk UniversityDepartment of Pathophysiology, Faculty of Medicine, Masaryk UniversityDepartment of Biochemistry, Faculty of Science, Masaryk UniversityAbstract 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.https://doi.org/10.1038/s41598-023-39575-8 |
| spellingShingle | Tereza Deissová Martina Zapletalová Lumír Kunovský Radek Kroupa Tomáš Grolich Zdeněk Kala Petra Bořilová Linhartová Jan Lochman 16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling Scientific Reports |
| title | 16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling |
| title_full | 16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling |
| title_fullStr | 16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling |
| title_full_unstemmed | 16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling |
| title_short | 16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling |
| title_sort | 16s rrna gene primer choice impacts off target amplification in human gastrointestinal tract biopsies and microbiome profiling |
| url | https://doi.org/10.1038/s41598-023-39575-8 |
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