4 Fastq files for a single run generated by 10X

Question

[hkarakurt]

Hello, I have a question about the 10X generated Fastq files. As I know 10X platforms can generate up to 4 Fastq files as R1, R2, I1 and I2. I need to use Fastq files and align them with CellRanger for a project. The data is coded as GSE154778 in NCBI GEO and PRJNA647411 in EBI ENA and NCBI SRA.

I downloaded the data using the code and metadata:

prefetch $LINE && /media/huk/PirLab/sratoolkit.2.11.0-ubuntu64/bin/fastq-dump --split-files --gzip ./$LINE

I have 4 Fastq files for some samples and 3 Fastq files for others. For example the SRR12273020 (https://trace.ncbi.nlm.nih.gov/Traces/index.html?view=run_browser&acc=SRR12273020&display=metadata) has 4 Fastq files and sizes are 230,8 MB-1,4 GB-383,6 MB and 310,9 MB And the SRR12273055 (https://trace.ncbi.nlm.nih.gov/Traces/?view=run_browser&acc=SRR12273055&display=metadata) has 3 Fastq files and sizes are 99,3 MB, 212,4 MB and 746,7 MB. How can I find which Fastq files are R1 and R2. As I know I1 and I2 reads are optional for CellRanger. Also, I can see single Fastq file for each sample in EBI ENA database.

Thank you in advance.

[Papyrus]

You can see the expected sizes in bp of the reads in the metadata links that you share. For one it is L=8, L=26, L=131 and for the other it is L=8, L=109, L=14, L=10. To confirm which is which, you can directly check your FASTQ files with head to see the length of the reads.

To then know which one is index, etc., this depends on the chemistry version. In your links it says 3' V2 for one of them, and that info is here, and for the other it says 3' V1; I've never used it but there's seems to be info around telling you which read is which, e.g. here (which also links to the v1 manual), telling you how 14 bp is i7 index, 8 bp is i5 index, etc..

Typically, the longest FASTQs (e.g. 131 bp, 109 bp) are the insert reads, because these are the only ones which sometimes are sequenced for more cycles than recommended.

[hkarakurt]

Thank you for your detailed answer. I will check the reads and compare them with chemistry manual as you suggested.

Thank you again for your help.