How to demultiplex an incomplete run using bcl2fastq2 and BCL Convert

How to demultiplex an incomplete run using bcl2fastq2

Add the use-bases-mask and ignore-missing flags to the bcl2fastq2 command to generate FastQ files from an incomplete run.

--use-bases-mask

Arguments:

• N or n - Ignore the cycle.

• Y or y - Use the cycle.

• I or i - Use the cycle for an Index Read

For example, if a run set up is 2x100 cycles with dual 10 bp index reads and the run aborted at cycle 50 of the second read, add the following flag to demultiplex the run:

--use-bases-mask Y*,I*,I*,Y49N*

In this example, all the cycles of Read 1, all the index cycles, and 49 cycles of Read2 are used while the rest of the cycles are masked out.

Note: use one fewer cycle than completed by the run.

In addition, add the following flags as well:

• --ignore-missing-bcls: The software ignores missing or corrupt BCL files and assumes 'N'/'#' for missing calls.

• --ignore-missing-filter: The software ignores missing or corrupt filter files and assumes that all clusters in tiles with missing filter files passed filter.

• --ignore-missing-positions: The software ignores missing or corrupt cluster location files. When cluster location files are missing, the software writes unique coordinate positions into the FASTQ header.

How to demultiplex an incomplete run BCL Convert

• Determine the last cycle sequenced.

• Create a new v2 sample sheet and use the OverrideCycles setting to mask the un-sequenced cycles using "N".

  1. For example, if a run was set up as 2x151 cycles with dual 8bp index reads and the last cycle sequenced was 177, set OverrideCycles to 151;I8;I8;Y10N141.

  2. For more information, see How to use the 'OverrideCycles' setting in the Run Planning User Interface.

• If the run aborted during the index cycles, use the OverrideCycles setting to mask the un-sequenced cycles using "N" and edit the sample sheet to remove the bases of the index that were not sequenced.

  1. For example, if a run was set up as 2x151 cycles with dual 8bp index reads and the last cycle sequenced was 162, set OverrideCycles to 151;I8;I3N5;N151 and remove the last 5 bases from the index2 column in the sample sheet.

• Upload the new sample sheet to a Project if using the BCL Convert app in BaseSpace.

• Requeue analysis using BCL Convert.

Note: BCL Convert, by default, has its strict mode turned off (--strict-mode false), so missing BCL/filter/BCI/locs files do not result in the analysis erroring nor ending prematurely.

For any feedback or questions regarding this article (Illumina Knowledge Article #3577), contact Illumina Technical Support [email protected].