Manifest and off target

Question

[KirGen]

Hello! I would like to understand better which kind of informations are contained in a manifest file? Are there only the genomic coordinates of the target regions?And how does it work the alinment against the reference genome? Maybe the genomic coordinates contained in the manifest are used to obtain the corrispondent sequences in the reference genome and against them will be done the alinment? And what about the % of off target,how can I detect it if it is removed during the analysis?How can I understand if I have a lot of off target?Because there there will be a low coverage? Thank you on advance!

Answer 1

[finswimmer]

Illumina's manifest file is a file format similar to bed. It describes region of interest in your analysis by defining genomic coordinates.

The alignment is independent of this file and should always be done against the whole reference genome. After this is done the variant calling is done only for the region listet in the manifest file.

The % of off-target reads is just a metric. Usually reads in this region are not removed. Your manifest file should list all region that where captured/enriched during library prep. A high % of off-target reads would indicate that the enrichment was not ideal.