I had some shotgun sequencing completed on DNA extracted from tissue biopsies with the intention on using them for metagneomic profiling. Each sample is paired end and was split over 4 lanes to maximise output on a NextSeq 500 2x150bp sequencing run.
I ran FastQC on fastq files and they all seem to be good qulity, however I am having issues merging the paired and fastq files. I initially combined all of the R1 and R2 files per sample using cat. I then tried using bbmerge to combine the merged R1 and R2 files as I have done previously, however the percentage of reads merging is only 10-15%, with the vast majority being ambiguous.
I have used this same workflow before on a previous project which was sequenced on a HiSeq using 2x126bp chemistry, and I was able to merge all of the samples with a succesfull rate of roughly 75% of the reads.
Am I doing something wrong? Should I expect differences based on the sequencing chemistry 2x150bp vs 2x126bp or the fact that the latest run was split over 4 lanes an issue?
Edit: This is the insert size info from BBmerge:
Insert range: 35 - 289 90th percentile: 278 75th percentile: 264 50th percentile: 236 25th percentile: 190 10th percentile: 135