Read numbers

Theory behind mothur
1

Hi all

I am characterizing the microbiome of the fish gut, using PE reads of the V4 region (Illumina MiSeq).

I have run 24 samples and get a total number of ~22m raw reads.

After the quality filtering and classification, I am left with anywhere from 100k-500k sequences per sample.

I know that this is a significant read depth to get to answer my question (are 12 control samples different from 12 treatment).

My question is this: In the output of classification the total number of reads are listed at the top of the table for each sample. However is this the total number of paired reads or single reads?

Also, is 100-500k reads per sample too deep, or does this matter?

Thank you in advance

2

The mothur outputs would be the number of contigs. So if you had 22m paired reads, you’d have 22m contigs, etc.

That does seem like a lot of sequences, but ultimately it depends on what you’re trying to do and your budget :slight_smile:

Pat

3

Thanks Pat

The aim is to determine if one group of 12 fish has a significantly different microbiome structure from another group of 12 fish.

Do you think that the read depth of 100k-500k quality filtered seqs per sample is too deep for this purpose or does this matter?

Thank you

4

I suspect it’s overkill.

Pat