Sequencing Error Rate

Commands in mothur
1

Hi all,

I was hoping to get a bit of guidance for addressing a potential sequencing error issue (bit of a broad question, I apologize)
I recently sequenced a number of samples and when I assess the error rate via seq.error, it’s quite high (~0.001, as opposed to the 6e-5 number cited in the Mothur SOP). I’m also getting ~100 OTUs for a sample that should only have 5 species, theoretically.

The batch file I used is based on the commands laid out in the Mothur SOP and I sequenced the V4 region using the V2 300 cycle kit. The samples were spiked onto a flow cell that was sequencing more than just 16s, if that matters.
I used a mock community I made myself, but all of the species were confirmed via sanger sequencing before hand.

I wasn’t sure if maybe I’m making the reference fasta wrong (I just copied the species-specific 16s fastas from NCBI and put them into one file) or if there might be some other issue with our pipeline. Is there anything that stands out that I should try to address first?

Any help is greatly appreciated!

Cheers,
Maria

2

The problem is probably that you are using the V3 chemistry. You really want to use the V2 chemistry. This post is still current…

http://blog.mothur.org/2014/09/11/Why-such-a-large-distance-matrix/

Beyond the bad chemistry, there are also problems with sequencing longer (i.e. 300 nt) than the fragment (i.e. ~275 nt). This will add to the error rate. I would strongly encourage you to go back and resequence using the 2x250 V2 chemistry.

Pat

3

Hi,
I have a follow-up question: is there a way to estimate sequence error rates other than the method with the mock community (described in the MiSeq SOP)? A reviewer to my paper asks for error rate though I have not performed mock-community analysis…
Yours,
Maxime

4

Nope. Sorry.

5

Hi Pat,
No, don’t be sorry! That’s what I had understood. I asked that question just in case…
Maxime