dada2 merging and comparing different data sets

Hello everyone,

I would like to compare two different data sets (ROMA1 and ROMA2) looking at the gut microbiome of patients with cancers undergoing various treatment regimens.

ROMA1 was sequenced using an Illumina MiSeq platform. The reads are 150bp PE . They used barcoded 515F and 806R primers and the V4 region was sequenced.

ROMA2 was sequenced using an Illumina MiSeq. The reads are 300bp PE . The 5155F (barcoded) and 806R primers were used to sequence the V4 region.

I ran the two data sets separately in dada2.

For ROMA1 I used the following filtering parameters

out <- filterAndTrim(fnFs, filtFs, fnRs, filtRs, truncLen=c(142, 140), maxN=0, maxEE=c(2,2), truncQ=2, rm.phix=TRUE, compress=TRUE, multithread= TRUE) # the primers were not in the data hence I didn’t need to remove

then I proceeded with the steps posted in the dada2 tutorial, removed chimeras and formed an otu table.

For ROMA2 I used the following filtering parameters

out <- filterAndTrim(fnFs, filtFs, fnRs, filtRs, trimLeft = 28, trimRight =20, truncLen=c(260, 220), maxN=0, maxEE=c(2,2), truncQ=2, rm.phix=TRUE, compress=TRUE, multithread= TRUE)

I then made my otu table for ROMA2.

Next I merged the OTU tables using mergeSequenceTables and then assigned taxonomy using the SILVAv138 database.

My questions/comments:

  1. Is it appropriate to compare the two data sets given these differences?

I tried cutting the ROMA2 reads to 142,140 to make them more comparable but had difficulties merging my forward and reverse reads.

  • I think it is appropriate as I am rarefying/normalizing my data to account for differences in sequencing depths of the two data sets but just wanted to confirm.
  1. Is it appropriate to merge ASVs belonging to the same species?

Any thoughts/tips would be much appreciated.

Thanks,
Alya

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