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Method Article
An optimized protocol for high-throughput amplicon-based microbiome profiling
Daryl M. Gohl
University of Minnesota Genomics Center, Minneapolis, MN, USA
Corresponding Author
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Here we describe an optimized protocol for amplicon-based microbiome profiling by next-generation sequencing (NGS). The protocol uses a modular, two-step PCR process that provides versatility as a common set of indexing primers can be paired with many sets of marker gene specific primers. We describe primer sets that have been used to prepare libraries for variable regions V1-V3, V3-V4 V3-V5, V4, V4-V6, and V5-V6 of the 16S ribosomal RNA (rRNA) gene, as well as for variable region V9 of the 18S rRNA gene and the ITS1 and ITS2 regions. This protocol is designed for high-throughput and incorporates the best practices for amplicon-based NGS library preparation that we have previously described. We have demonstrated that these best practices substantially increase accuracy compared to existing methods and also, importantly, minimize the dropout of taxa that have mismatches to the amplification primers.
Keywords
Microbiome, Next-generation sequencing, PCR, amplicon
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This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.xlsx
Table 2 Dual-indexing primers The sequence and suggested ordering layout of the indexing primer set (16 forward and 24 reverse primers, which allow multiplexing of up to 384 samples per lane) is shown, along with the indexing scheme (corresponding to the MiSeq setup templates), the index name, and the index sequence as it should appear in the sample sheet.
- supplement0.zip
Protocol Supplemental Files Supplemental Files
- supplement0.xlsx
Table 1 Marker gene primer sequences with dual-indexing adapters The marker gene targeted portion of the primer is highlighted in bold. * indicates a phosphorothiol bond, which prevents excessive adapter dimer formation with the ITS1 primer set.
Renuka
commented on 19 June, 2019
Does Table 2 refer to "indexing_primers.xlsx." If so, that only contains the primer sequences. There are no examples of plate layouts. Thank you.
Renuka
commented on 19 June, 2019
I believe there is a typo in Appendix 1, 1, iii: "Next transfer 20 μl of each forward index to the row(s) containing water." It should be "reverse index."
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Associated Publications
Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies
by Daryl M. Gohl, Pajau Vangay, John Garbe, +9
Nature Biotechnology (28 April, 2016)
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This is a preprint. It has not completed peer review.
Method Article
An optimized protocol for high-throughput amplicon-based microbiome profiling
Daryl M. Gohl
University of Minnesota Genomics Center, Minneapolis, MN, USA
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 25 Jul, 2016
Community comments: 2
Metrics
Comments: 2
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Here we describe an optimized protocol for amplicon-based microbiome profiling by next-generation sequencing (NGS). The protocol uses a modular, two-step PCR process that provides versatility as a common set of indexing primers can be paired with many sets of marker gene specific primers. We describe primer sets that have been used to prepare libraries for variable regions V1-V3, V3-V4 V3-V5, V4, V4-V6, and V5-V6 of the 16S ribosomal RNA (rRNA) gene, as well as for variable region V9 of the 18S rRNA gene and the ITS1 and ITS2 regions. This protocol is designed for high-throughput and incorporates the best practices for amplicon-based NGS library preparation that we have previously described. We have demonstrated that these best practices substantially increase accuracy compared to existing methods and also, importantly, minimize the dropout of taxa that have mismatches to the amplification primers.
Figure 1
Figure 2
Figure 3
Figure 4
Renuka
commented on 19 June, 2019
Does Table 2 refer to "indexing_primers.xlsx." If so, that only contains the primer sequences. There are no examples of plate layouts. Thank you.
Renuka
commented on 19 June, 2019
I believe there is a typo in Appendix 1, 1, iii: "Next transfer 20 μl of each forward index to the row(s) containing water." It should be "reverse index."
Associated Publications
Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies
by Daryl M. Gohl, Pajau Vangay, John Garbe, +9
Nature Biotechnology (28 April, 2016)