UMI workflow resulting in bams with empty reads

Question

[ww22runner]

Hello all,

In my NGS workflow for UMI based reads, I first tried identifying and removing sequence adapters using bbmerge and cutcadapt:

BBMERGE -Xmx1g -ignorejunk in1=SAMPLE_R1  in2=SAMPLE_R2 outa= adapters.fa itn
CUTADAPT -a forward_adapter -A reverse_adapter -o s_2_1_sequence_trimmed_UN.fastq.gz -p s_2_2_sequence_trimmed_UN.fastq.gz SAMPLE_R1 SAMPLE_R2

Then, I converted the trimmed fastq files to an unaligned bam:

java -Xmx4g -jar PICARD FastqToSam \
    O=SAMPLE_PFX_UN_unal.bam \
    F1=s_2_1_sequence_trimmed_UN.fastq.gz \
    F2=s_2_2_sequence_trimmed_UN.fastq.gz \
    SM=SAMPLE_PFX  \
    LB=null \
    PU=NA \
    PL=Illumina

and extracted the umi barcodes from these bams:

java -Xmx4g -jar FGBIO ExtractUmisFromBam \
    --input=SAMPLE_PFX_UN_unal.bam \
    --output=SAMPLE_PFX_UN_unal_umi_extracted.bam \
    --read-structure=5M2S+T 5M2S+T \
    --molecular-index-tags=ZA ZB \
    --single-tag=RX

When I use ValidateSamFile on the output produced above, I see that there are 8 empty reads in the bam:

ERROR:EMPTY_READ        8

which errors out when I try to perform the next step of converting the unaligned bam back to fastq:

  java -Xmx4g -jar PICARD SamToFastq \
        I=SAMPLE_PFX_UN_unal_umi_extracted.bam   \
        F=SAMPLE_PFX_UN_unal_umi_extracted.fastq \
        INTERLEAVE=true

The error seen:

Exception in thread "main" htsjdk.samtools.SAMFormatException: SAM validation error: ERROR: Record XXXXXXX, Read name XXXXXXX, Zero-length read without FZ, CS or CQ tag

It appears that removal of sequence adapters and UMI barcodes has resulted in empty reads that cause the error. Has anyone experienced this and are there any workarounds?

Thank you

Answer 1

[i.sudbery]

I don't know what the structure of your reads are - lots of UMI workflows lead to empty fastqs for one read or the other by design, as one read contains the barcdoes and nothing else.

However, whenever you trim adaptors, you must always be aware that you might create empty reads as they wer ebasically all adaptor. You can use the -m/--minimum-length switch to cutadapt to filter pairs with a read like this out. I would set it to filter out anything the same length as you UMI or shorter.

[ww22runner]

that makes sense, and worked, thank you!