After demultiplexing, most of the reads ended up in the ‘unknown’ bin. What could be the reasons for that?
The ‘unknown’ bin contains reads that couldn’t be matched to the specified index sequence or entire lanes that were not listed in the SampleSheet.csv file. The first place to check is the sample sheet and to verify that all the indexes were listed correctly and for the right lanes. Make sure that the number of index cycles in the sample sheet matches that in the config.xml file in the BaseCalls folder (or in the -qseq-mask parameter, if that was given to demultiplex.pl during the run). Finally, examine your read quality closely (in Status.xml) and see if the average basecall quality in the index cycles is low. This may indicate problems in the sample prep, or chemistry/instrument-related issues. If only a single cycle is suspected to be bad, try re-running demultiplex.pl with -mismatches 1.
The ‘unknown’ or ‘Undetermined_Indices’ bin contains reads that could not be matched to the specified index sequence or entire lanes that were not listed in the SampleSheet.csv file. First, check the sample sheet to verify that all of the indexes are listed correctly and are for the right lanes. Then, make sure that the number of index cycles in the sample sheet matches those listed in the config.xml file in the BaseCalls folder (or in the -qseq-mask parameter, if that was given to demultiplex.pl during the run). Finally, closely examine your read quality (in Status.xml) to see if the average basecall quality in the index cycles is low. This may indicate problems in the sample prep, or chemistry/instrument-related issues. If only a single cycle is suspected to be bad, try re-running demultiplex.pl with -mismatches 1.
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