Entering edit mode
3.4 years ago
goraadnan
•
0
Hi, I ran different 15 samples in a next seq 500 high output flow cell and got the sequencing result with good FATSQC report. But when I demultiplex using the barcodes (6bp) I found all the reads ending up in Undetermined.fastq (file size was around 90GB). The first thing that I did was to see if it was too much PhiX. So I mapped Undetermined.fastq to the PhiX genome and only 2.5% was mapped successfully. I am new in NGS. Can someone help me out of this issue?
Thanks in advance
Adnan
Are you the owner of this sequencer? If not you should ask the sequencing provider to do this for you. It would be easy for them and will save you a ton of headache.
If you are forced to deal with this yourself then please clarify if you have:
a) the full flowcell data folder (along with a working install of
bcl2fastq) ORb) just the "Undetermined" sequence files.
Depending on that there can be different answers.
I have a full flowcell data folder with very little of the reads demultiplexed as per sample sheet (although the file sizes are small, I can see all my samples in the folder) and a huge Undetermined file.
I actually had a total 30 samples, for which the libraries were prepared randomly and these were divided in two sets of 15 libraries each. This problem has occurred in only one of the runs (15 samples, file size of each is about 1.1Mb whereas the undetermined.fastq file size is 90GB) while the other run is perfectly fine (15 samples, file size of each sample was about 5Gb and the undetermined.fastq file is about 5Gb)
The indexes used were NEBNext oligos for (set 1, 2, 3 and 4) this was also done randomly.
Use the code contained in this answer: C: Demultiplexing reads with index present in the labels to identify the indexes that are present in your "Undetermined" reads. That should give you an idea of what may be happening. Perhaps you need to adjust your samplesheet and that should do the trick.