Hi all,
I've run 6 rounds multiplex PCR of 21 self-designed microsatellite primers on bear samples. I have pooled all the pcr products, purified the amplicons and sent for NGS sequencing.
I have Geneious software and also Linux workstation. Upon receiving the sequencing data, I hope the experts in ngs data analysis can help me with suggested workflow or commands to:
1. Isolate the sequence reads according to the microsatellite primers
2. Calculate the tandem repeats in each microsatellite loci sequenced in order to select suitable primers for genotyping of more samples
Thank you in advance.
Hi wai
as arthur said, multiplexing PCR without habving barcoding them means that at the end you have a pool of sequences, therefore impossible to address to samples.
but if you did it, you'll need first to align the sequences to the bear genome. from fastq files you'll get bam files, you can vizualize on some softs as IGV.
fred
1. You should be able to demultiplex the different PCRs, using the primers as your barcodes. There are a number of tools available for this. I've used scripts in QIIME (demultiplex_fasta.py or split_libraries_fastq.py) before, but there may be better standalone tools for your purpose.
2. Not my area of expertise, but quick googling suggests there are probably tools out there for that.
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