I am interested in doing paired-end 16S rRNA sequencing of the V4 region. The size of this region is about 251bp, I was advised by the sequencing company to use 150bp pared-end, however, does that mean that only about 50bp will overlap between the forward and the reverse reads...or possibly even less - because of the adapters at both ends? I would like to hear from others who have done this: personal thoughts, suggestions, recommendations. Thanks a lot!
Hi Stefanie,
in our group we routinely use the primers and protocols of the Earth Microbiome Project (https://earthmicrobiome.org/protocols-and-standards/16s/). It is a well established system for amplicon sequencing of the V4 region and you will have plenty of data and papers using it for comparison of your results.
Good luck,
René
Dear Stefanie,
In my opinion, suggestions from sequencing company is correct. Generally, V4 hyper-variable region is sequenced with 150 bp pair-end method using Illumina HiSeq platform and the same has been routinely adopted in plethora of previous research articles and megaproject such as the Earth Microbiome Project. Considering overlapping region, about 50 bp overlap between two reads will be sufficient in my knowledge as total read length is about 250 bp only. Moreover, during bioinformatics analysis you will be removing adapter and primer sequences.
You can also refer attached research article, which may help you.
As Badr recommended, 2x250 by Miseq platform is better for comparison or taxonomic classification. In this case, v3-v4 region is used. If you need only diversity analysis V4 region is enough.
Would you please help me understand how we decide which region of 16S for bacterian to go for? How much coverage we receive differs between different regions?
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