Information on leading r for UMIs when demultiplexing NextSeq 1000/2000 data with BCL Convert
When sequencing a run with Unique Molecular Identifier (UMI) situated in the index2 (i5) on a NextSeq1000/2000 instrument, BCL Convert will put a leading "r" in front of the reverse-complemented UMI in the FASTQ header. Downstream Illumina software solutions are aware and able to handle such case scenarios.
Example:
@VH00115:63:AACJFN3M5:1:1101:18534:1000:rAGACGCGCGTGCTGGGTCAGCGG 2:N:0:TTAGGC
BCL Convert will demultiplex indices according to the orientation in which the sequencer evaluated the index read(s). A flag in the RunInfo.xml specifies whether each index read was sequencing in the forward or reverse orientation. In the object of the RunInfo.xml, a IsReverseComplement flag will be specified as Y if the index read was sequenced in the reverse orientation, and as N if the index read was sequenced in the forward orientation.
BCL Convert will do the following when Y is specified for the IsReverseComplement flag for an index sequence:
- The software will reverse the sequence and generate the complement base pair as the index read, where A=T, C=G, and N=N
- The OverrideCycles value specified will be reversed for the corresponding index read before the reverse complement is taken
- If a UMI is specified in the corresponding index read, the r character will be added at the beginning of the UMI sequence written in the Read Name of the FASTQ file
- A log message will be displayed indicating that the reverse complement was used for the corresponding index read
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For any feedback or questions regarding this article (Illumina Knowledge Article #7945), contact Illumina Technical Support [email protected]. |