Question: Hello, I am trying to download fastq-files (**SRR12273024**) with fasterq-dump/fastq-dump from sra-tools. I have tried the --split-files and -s tags however, I only get 1 fastq file.

> @SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=109
> GATGTANAGAACGCGACTTCCACAAACCTGGATTTTTTATGTACAACCCTGACCCNGACCGTTTGCTATATTCCTTTTTCTATGAAATAATGTGAATGATAATAAAACA
> +SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=109 DDDDDI#< @SRR12273024.2 SN7001050R:482:HYKG3BCXX:1:1101:1096:2166 length=109
> AAGGTACCTGGGTTCAACTAAAGCGCCAGCCTGCTCCACCCAGAGAAGCACACTTTGTGAGAACCAATGGGAAGGAGCCTGAGCTGCTGGAACCTATTCCCTATGAATT
> +SRR12273024.2 SN7001050R:482:HYKG3BCXX:1:1101:1096:2166 length=109 DDDDDIHIIIHIFHIIIIIIIIIIGIHIIIIIIIIIHIHHHIGHIHIHI?GHHG?GFHHDH@FG< @SRR12273024.3 SN7001050R:482:HYKG3BCXX:1:1101:1086:2183 length=109
> CAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

I have tried various ways to de-interleave the fastq file including methods outlined in: gist.github.com/nathanhaigh/3521724 and
biostars.org/p/19446/
however, none of these methods output fastq files that are compatible with the cell ranger pipeline.

When I try to run cellranger counts on the file, I am given this error:

> Log message: The read lengths are incompatible with all the
> chemistries for Sample SRR12273024 in ./
> - read1 median length = 109
> - read2 median length = 0
> - index1 median length = 0
>
> The minimum read length for different chemistries are: SC5P-R2 -
> read1: 26, read2: 25, index1: 0 SC5P-PE - read1: 81, read2: 25,
> index1: 0 SC3Pv1 - read1: 25, read2: 10, index1: 14 SC3Pv2 -
> read1: 26, read2: 25, index1: 0 SC3Pv3 - read1: 26, read2: 25,
> index1: 0

>We expect that at least 50% of the reads exceed the minimum length.

I've looked into this error and it seems like the dataset is paired-end, which is why I have been trying to split the files using sra-tools to no avail.

Any help is appreciated!

Answer: Seems there is one read but you can split the splots to get four different reads, being the index (1), the cDNA (2) and then the Cellular Barcode + UMI (3+4) which matches the description of the old v1 chemistry here at **(8) Final library structure**.

https://teichlab.github.io/scg_lib_structs/methods_html/10xChromium3v1.html

$ fasterq-dump SRR12273024 --split-spot --include-technical --split-files

$ head -n 4 SRR12273024*
==> SRR12273024_1.fastq <==
@SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=8
GCCAACAA
+SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=8
DDDDDHII

==> SRR12273024_2.fastq <==
@SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=109
GATGTANAGAACGCGACTTCCACAAACCTGGATTTTTTATGTACAACCCTGACCCNGACCGTTTGCTATATTCCTTTTTCTATGAAATAATGTGAATGATAATAAAACA
+SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=109
DDDDDI#< SRR12273024_3.fastq <==
@SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=14
GCACGTCTTATTCC
+SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=14
@?D?DDE@GC?11<

==> SRR12273024_4.fastq <==
@SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=10
CGTGCCACAG
+SRR12273024.1 SN7001050R:482:HYKG3BCXX:1:1101:1163:2092 length=10
DDDDDHIIII

Not sure how to get that into CellRanger format though, never used CellRanger and V1 chemistry myself. Others may know, or you can check the CellRanger manual for the expected file (formats). Does it expect two or three (or even all four) files?