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We will be out of the office for a Broad Institute event from Dec 10th to Dec 11th 2019. We will be back to monitor the GATK forum on Dec 12th 2019. In the meantime we encourage you to help out other community members with their queries.
Thank you for your patience!
NextSeq 500 Lanes and BaseRecalibrator
I am working with RNA-seq data from the NextSeq500: 144 samples, with 24 samples multiplexed into an equi-molar pool for each of the 6 runs.
The NextSeq flowcell consists of 4 lanes that are supplied from from a single reservoir, so the same pool must be sequenced on all 4 lanes. On other platforms such as the HiSeq, the 8 lanes have to physically be loaded separately even if they are sequencing the same pool.
My understanding is that BaseRecalibrator should be run for each lane of data, which I can specify using the PU read groups tag. My sequencing provider de-multiplexed the raw FASTQ reads by sample, but not by lane, so I currently have 144 BAM files, one BAM file per sample, which contain mapped reads (using STAR) for that sample sourced from all 4 lanes of the run. In order to assign different PU read group tags to reads from different lanes using Picard, I would need to split either the BAM file or the raw FASTQ files by lane, then process the 4 files separately.
Should I be treating each of these 4 NextSeq lanes separately like HiSeq lanes, or can I run BaseRecalibrator on all 4 lanes together since the lanes are supplied from one reservoir?