Forward and reverse single cell data analysis
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6 weeks ago
elb ▴ 200

Hi, I have to analyze published scRNA-Seq data. Data are Chromium 10X on NovaSeq6000 and: for each sample I have forward (R1) in lane1 and lane2 (and hence two files) and reverse (R2) in lane1 and lane2 (and hence two files). Totally I have 4 files for sample. I have .fastq files. I usually analyze 10X scRNA-Seq data using Cellranger in single strand starting from the demultiplexing and ending with the alignment. It is the first time I have forward and reverse fastq files on multiple lanes and I don't know how to analyse them. Can anyone give me some hints on the analysis I have to do? Suggestions on tutorials, tools, papers about my task are more than welcome.

Thank you in advance

RNA-Seq • 142 views
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Are you still planning to use cellranger?

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No no it is the practice of the lab but it is not mandatory

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6 weeks ago
ATpoint 47k

Just use cat to merge the R1s and the R2s respectively.

cat R1_lane1.fastq.gz R1_lane2.fastq.gz > R1.fastq.gz

same for R2.

See answer from swbarnes2 below, apparently CellRanger is picky about file names. I am not a CellRanger user, I prefer lightweight approaches such as Alevin for scRNA-seq quantification and for this (and probably most other applications) cating is fine. A CellRanger-like approach that is faster and more generic (=less picky) would be STARsolo.

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I think you meant "zcat"

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cat works as well.

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No, zcat would decompress the stream which is unintended. cat is well able to concatenate two gzip-compressed files producing a single gzip-compressed file.

 echo 123 | gzip > foo.txt.gz
 echo 456 | gzip > bar.txt.gz
 cat foo.txt.gz bar.txt.gz | gzip -dc
 123
 456
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That's great. Good to know and thanks for the tip.

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That will not work. Cellranger is fussy about file names, it won't read that file in.

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#til -- another reason to use Alevin over CellRanger :-P

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6 weeks ago
swbarnes2 9.7k

Don't cat the files. Cellranger is smart, it will get files with the same sample name from all lanes.

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