Cellranger count error: Input FASTQ file ended prematurely

Question

[bp22]

Hi all,

I am trying to align and produce counts form some scRNA-seq using CellRanger 7.1.0. The fastq files for input are of the following format (for illustration):

SI-GA-A1_1_S1_L001_I1_001.fastq.gz
SI-GA-A1_1_S1_L001_R1_001.fastq.gz
SI-GA-A1_1_S1_L001_R2_001.fastq.gz

SI-GA-A1_2_S2_L001_I1_001.fastq.gz
SI-GA-A1_2_S2_L001_R1_001.fastq.gz
SI-GA-A1_2_S2_L001_R2_001.fastq.gz

SI-GA-A1_3_S3_L001_I1_001.fastq.gz
SI-GA-A1_3_S3_L001_R1_001.fastq.gz
SI-GA-A1_3_S3_L001_R2_001.fastq.gz

SI-GA-A1_4_S4_L001_I1_001.fastq.gz
SI-GA-A1_4_S4_L001_R1_001.fastq.gz
SI-GA-A1_4_S4_L001_R2_001.fastq.gz

and these fastq files are from the same sample. I am using the following cellranger count options:

cellranger count --id=EXP20_CELLRAN7_1_0 \
--fastqs=/folder1/fastq_folder \
--sample=SI-GA-A1_1,SI-GA-A1_2,SI-GA-A1_3,SI-GA-A1_4 \
--transcriptome=/refdata-gex-GRCh38-2020-A

However, I am encountering the following issue:

14: start_thread
15: clone
Input FASTQ file ended prematurely: file: "/SI-GA-A1_4_S4_L001_R1_001.fastq.gz", line: 153386036

I have aligned the same set of fastq files using STAR solo and it ran successfully. Hence, I am not able to understand what might be the issue here. Any help is highly appreciated.

Thank you.

Best, BP

[GenoMax]

It would be prudent to validate that the fastq file is not corrupt: https://github.com/biopet/validatefastq

[bp22]

Dear GenoMax

Thanks for your suggestion.

Indeed! I have validated the fastq files using the tool that you have mentioned and there doesn't seem to be anything wrong with the fastq files:

INFO  [2023-07-27 17:03:05,734] [ValidateFastq$] - Possible quality encodings found: Sanger, Illumina 1.8+
INFO  [2023-07-27 17:03:05,734] [ValidateFastq$] - Done processing 81652310 fastq records, no errors found
INFO  [2023-07-27 17:03:05,734] [ValidateFastq$] - Done

Thank you.

Best, BP

[GenoMax]

Have you tried re-running cellranger? Perhaps that was a temp glitch of some sort.

Not directly related but It is also a bit odd to name sample fastq files with 10x library codes. Were these files made using indexes listed on separate lines (e.g. 4 that go with SI-GA-A1) on each line in the samplesheet during demultiplexing?

[bp22]

Thanks for the suggestion! Yes, I have tried re-running cellranger and I have tried different versions of cellranger as well. I ran into the same issue.

Not directly related but It is also a bit odd to name sample fastq files with 10x library codes. Were these files made using indexes listed on separate lines (e.g. 4 that go with SI-GA-A1) on each line in the samplesheet during demultiplexing?

I am not aware of the demultiplexing procedure as the data was sent to us from a sequencing facility long back. However, I am aware that the fastq files are from the same sample and I have the one of the scenarios as described here by 10X: Specifying input 10X

under the section:

My FASTQs are in an output folder from mkfastq or bcl2fastq, but there are multiple folders per sample index, like "SI-GA-A1_1" and "SI-GA-A1_2"

Do you think it would be appropriate to concatenate the fastq files into one and then run cellranger pipeline given that the fastq files are from the same sample and same lane?

[swbarnes2]

If _1 and _2 really are the same library, you should rename the fastqs, or make symlinks, such that they have the same sample name, but different lanes. Cellranger will naturally take them all in together.

Answer 1

[bp22]

Dear all,

The reason for the error was due to the index (I1) fastq files, for example SI-GA-A1_1_S1_L001_I1_001.fastq.gz having a lot more entries than both R1 and R2 fastq files. In principle, all three fast files should have the same number of line counts. Ignoring the index file during the CellRanger run solved the issue.

Thank you.

[GenoMax]

That is odd. Thanks for following-up and providing an explanation.

[ATpoint]

Just delete the index read files. They just take up space and will never be used.