Hi. I am currently doing targeted sequencing. We targeted 400genes with depth of 500. I just entered the field of bioinformatics and this is my first project. I am wondering if there is well recognized practice for targeted sequencing analysis tool options/pipelines. There are handful of choices for exome sequencing analysis tools, but for targeted, I having hard time to find reliable information/sources.
If you have any suggestions or prior experiences in targeted sequencing i would like to hear your opinions.
Thank you.
1 answer
How was the target enrichment performed? Using capture probes (similar to exome sequencing) or multiplex PCR?
(Try to be as informative as possible when asking questions!)
If capture probes:
Process exactly like exome sequencing, see: GATK Best Practices
If multiplex PCR:
Process almost exactly like exome sequencing (see above) with two exceptions:
- Don't remove/mark duplicates reads
- No variant calling in the primer sequences
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Hi Thank you so much for your help. My apologies for lack of information about our set up. We used capture probes.
Can i ask you one more questions?
1) Is there customized or optimized filtering options and tools for targeted sequencing analysis. Our sample read depth is 500x in average.
Thank you for your help.
Please use
ADD COMMENTto reply to earlier answers, as such this thread remains logically structured and easy to follow.You mean variant filtering?
Unlike exome sequencing, your dataset may be too small to apply VQSR. In that case you should use hard filtering. On the GATK website you can find more material for further reading, including Understanding and adapting the generic hard-filtering recommendations.
Thank you so much for your answers. I deeply appreciate it. Thank you.