rRNA bacterial gene and fungal ITS metagenomics samples

Introduction

The GSAF uses a design based on this tech note from Illumina for reading of the V4/V5 bacterial 16S rRNA gene region, and has extended it to other useful metagenomics primers.  This design is a nested PCR in which the first (inner) PCR primes the gene-specific region and adds the Illumina sequencing primer sites, then the second (outer) PCR adds Illumina indexes and flow cell binding/amplification sites.  The Illumina sequences are "Nextera" not "TruSeq".

rRNA gene-specific primer sequences are shown in this teal color. 

Illumina platform-specific sequences are shown in this green color.

Dual-barcode sequences are shown in this orange color.

NOTE that all primer pairs are shown here FWD: 5'-3', REV: 3'-5'

Bacterial 16S rRNA V1/V2 primers (version 1):

Inner (first) round:

Hyb8F_rRNA: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTTTGATCMTGGCTCAG -3'

Hyb338R_rRNA: 3'-TGAGGATGCCCTCCGTGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-5'

Hyb8F is a 3 bp truncation of the classic 27F primer and has been used in many NGS studies such as this one.  Hyb338R is a 3 bp truncation of the classic 338R primer.  These primers amplify a 343 bp region in E. coli.

Bacterial 16S rRNA V4/V5 primers  (version 1) - these amplify Eukaryotic 18S rRNA!!!:

Inner (first) round:

Hyb515F_rRNA: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTA -3'

Hyb909R_rRNA: 3'-TGARTTTMCTTAACYGCCCCGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-5'

These are based on, "Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies" by Yong Wang and Pei-Yuan Qian, 2009.

 In E. coli (O157:H7) these primers amplify a total region of 414 bp from the 16S gene with the final sequencing library being exactly 485 bp.

Barcoding primers, common to all designs:

Outer (second) round primers (examples of two specific indexes are shown, both 5'->3'):

Hyb_F01_i5, AATGATACGGCGACCACCGAGATCTACAC ATCACG TCGTCGGCAGCGTC

Hyb_R21_i7, CAAGCAGAAGACGGCATACGAGAT CGAAAC GTCTCGTGGGCTCGG

Information on universal primers (515/926R) for those who would like to use them and submit amplicons to the core (thanks Daniel Acosta for sharing)

• https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.15553
• https://journals.asm.org/doi/10.1128/mSystems.00565-21
• https://github.com/jcmcnch/eASV-pipeline-for-515Y-926R
• https://www.protocols.io/private/C0F9404AB3DAEC96683F142351CEF59C

This paper is an excellent resource from the SILVA group on Bacteria, Archaea, and Eukaryota.  Here is a link directly to their main supplemental table of primer designs and coverage.