Thank You so very much. I wanted to discuss one more thing with you. In my paired-end sequences, the forward and reverse reads does not match, it means they are not in the same order. and I have read in the qiime2 documentation that qiime2 and dada2 need ordered paired-end reads but in tutorial example the two sequences forward.fastq.gz and reverse.fastq.gz are also not in the same order but still giving the good level taxanomy. I am confused. I have checked reads order using this R script. # Load forward and reverse reads
fwd_reads <- readFastq("D:/Local Disk D_71520222153/Desktop/metagenomics-knut/beet/beet/forward.fastq.gz")
rev_reads <- readFastq("D:/Local Disk D_71520222153/Desktop/metagenomics-knut/beet/beet/reverse.fastq.gz")
Extract sequence IDs (headers without @ symbol)
fwd_ids <- as.character(ShortRead::id(fwd_reads))
fwd_ids
rev_ids <- as.character(ShortRead::id(rev_reads))
Find IDs in forward reads but not in reverse reads
missing_in_rev <- setdiff(fwd_ids, rev_ids)
missing_in_rev
This script showed so many sequences that are in forward sequence but not in the reverse sequence file. same is the case with my own sample sequences file as well but still this is giving good taxanomy and mine samples are giving poor taxanomy using same qiime2 pipeline. Please help me with this confusion. I would be very thankful to you