Hi there,
Recently, we made PE Illumina sequencing of multiplexed libraries (~320 bp and up to 23 libraries per lane). Target libraries were prepared with single 6-bp Illumina index (not dual), and the cluster was generated using PE cluster kit v3.
Unfortunately, during the sequencing, the technician did not specified about indexing. Now I generate sample sheet (describing the samples and indexes) and tried to demultiplex and regenerate Fastq files with Cassava 1.8.2. But the majority of the reads were in ‘unspecified indices’ directory. I tried to adjust –use-base-mask option, but no changes. The other problem I had with –use-base-mask setting is that the length of each read is 104 bp (R1 & R1 = 204, which is also specified in both Config & RunInfo files), and when I set –use-base-mask as Y*n,I6n,Y*n or Y*,I6,Y*, it set the index containing reads to be R2, but still no changes. I am not sure if my –use-base-mask setting is right. When I run Cassava with default settings, error message saying ‘expected barcode length –including delimiters is 0’ and the program terminated. Please any helps and suggestions on how to fix this?
I also tried other three tools – Fastx, EA-utils and Sabre – but they are not good enough (majority of the reads without index).
I enclosed the top-reads generated from the original settings (without indexing).
Thanks!
Recently, we made PE Illumina sequencing of multiplexed libraries (~320 bp and up to 23 libraries per lane). Target libraries were prepared with single 6-bp Illumina index (not dual), and the cluster was generated using PE cluster kit v3.
Unfortunately, during the sequencing, the technician did not specified about indexing. Now I generate sample sheet (describing the samples and indexes) and tried to demultiplex and regenerate Fastq files with Cassava 1.8.2. But the majority of the reads were in ‘unspecified indices’ directory. I tried to adjust –use-base-mask option, but no changes. The other problem I had with –use-base-mask setting is that the length of each read is 104 bp (R1 & R1 = 204, which is also specified in both Config & RunInfo files), and when I set –use-base-mask as Y*n,I6n,Y*n or Y*,I6,Y*, it set the index containing reads to be R2, but still no changes. I am not sure if my –use-base-mask setting is right. When I run Cassava with default settings, error message saying ‘expected barcode length –including delimiters is 0’ and the program terminated. Please any helps and suggestions on how to fix this?
I also tried other three tools – Fastx, EA-utils and Sabre – but they are not good enough (majority of the reads without index).
I enclosed the top-reads generated from the original settings (without indexing).
Thanks!
Comment