Hello,
This may not be related to bioinformatics, but I demultiplexed one illumina sequence run and noticed that one sample has quite different the # of reads from different lanes. (used the same index sequence in the samplesheet)
sample1 from lane1,3,4 : 0 reads
sample1 from lane2 : 8 million reads.
Would you mind explaining why it happened? It's nanostring sequence data. If you need more info about the libraries, I will definitely ask questions to my coworkers who prepared the libraries and ran the sequencer.
Which sequencer is this from? If the same pool did not run on multiple lanes then this would be the observation. Perhaps you received/created a wrong samplesheet. I suspect this to be case since there are 0 reads in other lanes.
Based on 4 lanes this is likely NovaSeq 6000, which can be run in XP mode where 4 separate pools can go on 4 lanes (or you could load the same pool 1 to 4 times).
Yes, you are correct. Novaseq instrument with S4 flowcell. I think I heard that XP mode was used. Yes, I will look into the samplesheet again.
So there could be possiblities that 4 seprate pools have something to do with the # of reads? (Sorry, I'm not familar with web lab work for the sequencers) even if I have a correct samplesheet?
Thank you for your insight!
If a single pool is loaded on all 4 lanes then one can see differing number of reads for any samples in that pool. But they will still be in the ballpark (within a few thousand) across the 4 lanes. No sample would have 0 reads in one or more lanes and 8 million in one lane.
If completely independent pools are loaded on 4 lanes then you would not expect the samples in a specific pool to show up on other lanes ... unless other lanes have samples that share the same index. Good bookkeeping is a MUST with sample pools since this information is being used to create the samplesheet.
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