Hello, im new here and i have a question about a sequencing problem we were getting.
For a project, we use Miseq and nano kit for doing 2*151 paired sequencing.
We sequence 1 or 2 amplicons of known coding with 1 snp of interest.
in each run we sequence 3 o 4 biological samples as duplicates or triplicates for a total 13 barcodes of 6 bp).
The design is made this way to get a 100% overlap between R1 and R2 cause we need accuracy calling the SNP (the original sample has a very low dna suppossed to carry the snp and high quantity of DNA know to be WT).
Important to say that basically on the PCR to add the adapters and barcodes the amplified sequence is only 2 bp (including the snp position). And we use 20% phix to compensate the low variability.
The problem:
For one of the amplicons (runing alone or with the other amplicon) everything is okay- A little low R2 quality but fine. also, cause the insert size is lower than 151 we get some low quality poly A and the end of the reads but thats not a problem.
For the other amplicon (another gene) the forward read is OK but the reverse read is a nightmare. We don't know whats happening.
Attached is the FastQC report for 1 amplicon_good and amplicon_bad R1 and R2, from the same run.
Thankyou
For a project, we use Miseq and nano kit for doing 2*151 paired sequencing.
We sequence 1 or 2 amplicons of known coding with 1 snp of interest.
in each run we sequence 3 o 4 biological samples as duplicates or triplicates for a total 13 barcodes of 6 bp).
The design is made this way to get a 100% overlap between R1 and R2 cause we need accuracy calling the SNP (the original sample has a very low dna suppossed to carry the snp and high quantity of DNA know to be WT).
Important to say that basically on the PCR to add the adapters and barcodes the amplified sequence is only 2 bp (including the snp position). And we use 20% phix to compensate the low variability.
The problem:
For one of the amplicons (runing alone or with the other amplicon) everything is okay- A little low R2 quality but fine. also, cause the insert size is lower than 151 we get some low quality poly A and the end of the reads but thats not a problem.
For the other amplicon (another gene) the forward read is OK but the reverse read is a nightmare. We don't know whats happening.
Attached is the FastQC report for 1 amplicon_good and amplicon_bad R1 and R2, from the same run.
Thankyou
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