Hello everyone!
I am totally new in NGS, so I apologize for the basic question.
I am doing ddRAD (EcoRI and SphI) for SNP analysis in a fish species with genome size ~900 Mb.
I got my raw data from the MiSeq and found out that the distribution of reads varies a lot among individuals (each individual is identified with a barcode) (the image attached shows total number of reads -2nd column- for each barcode -first column- after running "process_radtags" in STACKS, used sliding window 0.1 and minimum quality score 20). Some libraries got 11M reads whereas others got ~8000, no good.
I wonder, what else, other than an incorrect normalization of the DNA concentration when I started the protocol, may explain this outcome? Maybe the barcodes I used were not diverse enough among samples within the same sequencing run? A side note, I used 5% PhiX. For DNA quantification I used Quanti-PicoGreen
Thanks in advance for any advice!
I am totally new in NGS, so I apologize for the basic question.
I am doing ddRAD (EcoRI and SphI) for SNP analysis in a fish species with genome size ~900 Mb.
I got my raw data from the MiSeq and found out that the distribution of reads varies a lot among individuals (each individual is identified with a barcode) (the image attached shows total number of reads -2nd column- for each barcode -first column- after running "process_radtags" in STACKS, used sliding window 0.1 and minimum quality score 20). Some libraries got 11M reads whereas others got ~8000, no good.
I wonder, what else, other than an incorrect normalization of the DNA concentration when I started the protocol, may explain this outcome? Maybe the barcodes I used were not diverse enough among samples within the same sequencing run? A side note, I used 5% PhiX. For DNA quantification I used Quanti-PicoGreen
Thanks in advance for any advice!
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