Hi,
I ran a library on the Illumina MiSeq at our institute with the V2 Kit (2 x 250 bp) and got rather bad read quality. The library contained 16S rRNA amplicons, the v4 region, of 79 fish skin samples. We did the dual indexing strategy described by Kozich et al. 2013.
The average quality is not too bad (73.9 % >=Q30) but the heatmaps looks terrible. I attached you the detailed information.
Normally I use USEARCH for merging the pairs according to quality check and when trying this I get empty output files and it tells me all my reverse reads are too short. Even when changing the parameters to a minimum of strictness the reverse read goes to the trash.
After usearch I would have followed the mothur pipeline. Interestingly, mothur merges my pairs since their algorithm is not very strict. However, I don't trust my results at all.
I'm in contact with some MiSeq experts and they rather assume the library itsself to be the problem (or the DNA) than the seqeuncing run.
Since I had a lot of trouble with getting nice PCR bands it could be the library (but what can have caused problems?) and I have to deal with the bad output.
Now, I'm thinking of using only the forward read for the analysis but I might loose a couple of nts at the end.
Did anybody of you had some similar sequencing output and give me some hints how I could improve the quality? Maybe I have to increase the diversity? We spiked in 10% phiX.
Thanks a lot!
I ran a library on the Illumina MiSeq at our institute with the V2 Kit (2 x 250 bp) and got rather bad read quality. The library contained 16S rRNA amplicons, the v4 region, of 79 fish skin samples. We did the dual indexing strategy described by Kozich et al. 2013.
The average quality is not too bad (73.9 % >=Q30) but the heatmaps looks terrible. I attached you the detailed information.
Normally I use USEARCH for merging the pairs according to quality check and when trying this I get empty output files and it tells me all my reverse reads are too short. Even when changing the parameters to a minimum of strictness the reverse read goes to the trash.
After usearch I would have followed the mothur pipeline. Interestingly, mothur merges my pairs since their algorithm is not very strict. However, I don't trust my results at all.
I'm in contact with some MiSeq experts and they rather assume the library itsself to be the problem (or the DNA) than the seqeuncing run.
Since I had a lot of trouble with getting nice PCR bands it could be the library (but what can have caused problems?) and I have to deal with the bad output.
Now, I'm thinking of using only the forward read for the analysis but I might loose a couple of nts at the end.
Did anybody of you had some similar sequencing output and give me some hints how I could improve the quality? Maybe I have to increase the diversity? We spiked in 10% phiX.
Thanks a lot!
Comment