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

For any feedback or questions regarding this article (Illumina Knowledge Article #7945), contact Illumina Technical Support [e-mail].