Infer UMI from fastq files

Question

[Ar]

I have RNA-Seq from Illumina NextSeq1k2k using NEB E7416 Unique Dual Index UMI Adaptors RNA Set, where the UMI are added on the i7 index. The bcl2fastq (v4.2.7) was used to generate the fastqs using the following settings:

The generated fastq looks good. I have a question regarding the UMI in the header of each read. I am interested in knowing if the bcl2fastq produces the UMI in the 8th column of the header of the read. For example:

zcat 01_Cond1_R1_001.fastq.gz | head -8

@VH01090:243:AAFNG5MM5:1:1101:40462:1000:TAGGAGTATGG 1:N:0:GTACACCT+CATGAGGA GNTTCACCGGCGGCCCGCAGGGCCGGCGGACCCCGCCCCGGGCCCCTCGCGGGGACACCGGGGGGGCGCCGGGGGCCTCCCACTTATTCTACACCTCTCAT + C#CCCCCCCCCCC;CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC;CCCCCCCC-CCCCCCCCCCCCCCCCCCCCCCC;C

Is TAGGAGTATGG the UMI?

@VH01090:243:AAFNG5MM5:1:1101:41030:1000:CCCTGCTATAA 1:N:0:GTACACCT+CATGAGGA CNTAGGATAATAGCGCTTTGTTGTCTCTCCTGCCACAGGAAGGCTCCATGGTTGTCCTACTTTCAGCCTTCGGTGCCTTTAGTGAGGGGTACCTGAAAAAT + C#CCCCCCC;C;C-CCCCCCCCC;CCCCCC;CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC-CCCCCCCCCCCCCCCCCC;CCCCCCCCCCCCCCCCCC

Is CCCTGCTATAA the UMI?

Answer 1

[GenoMax]

bcl2fastq (v4.2.7) was used to generate the fastqs

Based on the software version number you must have used bcl-convert rather than bcl2fastq.

But in either case following should be true.

Is CCCTGCTATAA the UMI?

As noted on page 17 of bcl2fastq guide: https://support.illumina.com/content/dam/illumina-support/documents/documentation/software_documentation/bcl2fastq/bcl2fastq2-v2-20-software-guide-15051736-03.pdf that is the UMI.

[Ar]

Thank you for the confirmation. Yes, you are right. It is bcl-convert