10X scRNA-seq v2 odd fastq format

Question

[Dave Carlson]

Hi Biostars,

I've downloaded some scRNA-Seq data from the GSA, which I am hoping to analyze. This is 10x V2 chemistry sequence data.

However the format is different from what I am familiar with. First, the reads come in 2 fastq files ("f1" and "r2"):

CRR034505_f1.fastq.gz
CRR034505_r2.fastq.gz

More importantly, both mates have equal read lengths:

zcat CRR034505_f1.fastq.gz | head -n4
@ST-E00126:655:HL5FTCCXY:5:1101:7638:1151 1:N:0:NAAGTGCT
NAGTAACCAAGACACGTATTGCGCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAACTAAAAAGGGGTCCCAGAATTTCAGCAGTTCTCTGATTTTTATATTTTATTCCTCTTCCTATCCAATCCCTGCCTTTTGCTTCAAGGTG
+
#AAFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFAA--7<<--AFFJ7-)-7)--<AAF7<<--7-----77---AA7AFJF7F<A----7A-<-AA<F<A7-A-77)--<F-<<--7A--<-77

zcat CRR034505_r2.fastq.gz | head -n4
@ST-E00126:655:HL5FTCCXY:5:1101:7638:1151 2:N:0:NAAGTGCT
NCAAAGAAAAAGACACATTTGGGAAGAAAAGCAGGAAAAACGTTAAAGAAAATGTACTTACCACCTGGACTCAAAAGGCAGGGATTGGATAGGAAGAGGAATAAAATATAAAAATCAGAGAACTGCTGAAATTATGTGACCACTTTTTAG
+
#AAFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJAJJJJJJJJJJJJJJJJJJJJJJJJFJFJJJJJJJJJJJJJJAJJJJFJJFAJFJJJJJJJJJJJJJJJJJJF-A7JAAF---7<<AFJFF<AJ--F-)-7<<FJAFA

I have never seen a scRNA-Seq data set that looks like this, though I have not done much work with 10x V2 chemistry in the past.

Is this normal? Has anybody encountered scRNA-Seq data like this before?

Thanks!

Dave

Answer 1

[GenoMax]

Some people sequence 10x libraries for equal length R1/R2 ignoring 10x sequencing recommendations. This is probably done for convenience of the sequencing centers. You can either choose the first 26 bp from read 1 (as you can see it reads) since at that point cell barcode + UMI is followed by the poly-dT linker. If you are planning to use cellranger it should take the bases it needs from R1 file.

*NAGTAACCAAGACACGTATTGCGCAT*TTTTTTTTTTTTTTTTTTTTTT

[Dave Carlson]

Fantastic, thank you!

[Ming Tommy Tang]

just make sure you compare the R1 reads after deleting the other bases to the 10x whitelist to make sure those are cell barcodes. R1 is the 16bp feature barcode + 10 bp UMI https://divingintogeneticsandgenomics.com/post/understand-10x-scrnaseq-and-scatac-fastqs/ v3 is 12bp UMI if I recall.