Explanation of SQuIRE Count output file format

[Winbuntu]

Hi I wonder if you could add explanations about the format of the files generated by SQuIRE Count. This would be helpful for people who want to perform customized analysis besides differential expression on their own.

I think a good example of format explanations is from StringTie: http://ccb.jhu.edu/software/stringtie/index.shtml?t=manual#output

Specifically for me, I am wondering what each column in *_refGenecounts.txt means. For example,

chr17 26138685 26195811 Axin1 11.196273 + 264 NM_001159598,NM_009733

I think 11.196273 is the TPM, and 264 is the read count? And how did you deal with multiple mapped reads that align to genes?

Thanks.

[Winbuntu]

Also, it would be helpful if you could clarify that each column means in *_TEcounts.txt file.
i.e. if "fpkm" computed using "uniq_counts" , "tot_counts" or "tot_reads"?

[cpacyna]

Hello,

Sorry for the long response time. Thanks for your feedback; we'll work on building documentation of the output files.

For *_refGenecounts.txt, here are the headers:
chr: chromosome of transcription
tx_start: coordinate for start of transcription
tx_stop: coordinate for end of transcription
Gene_ID: gene name
fpkm: fragments per kilobase per million reads
strand: + or - for stranded data
count: read count, computed by transcript length * coverage / readlength
transcript_ID: transcript specifier

We used the base StringTie parameters for gene expression counting (-M .95) for guided assembly.

We're working on making the full *_TEcounts.txt format documentation, but for the time being, fpkm is computed using total counts.

Thanks for your patience as we sort it out! Let us know if you have other questions in the meantime.

Regards,
Chloe

[bvaldebenitom]

Hi there!

Has this been sorted out? I'm running some analysis on these files, and need a bit of clarification in the *_TEcounts.txt headers.

[MaxwellShih]

Hello,
I am interested in getting the TPM (transcripts per kilobase million) for TE, which is lacking in the version I am using. I am trying to calculate the TPM from fpkm. (https://haroldpimentel.wordpress.com/2014/05/08/what-the-fpkm-a-review-rna-seq-expression-units/?fbclid=IwAR0gVbmAYOZzIKWtQJU8SHUbQ5Nhh4NaSIi9ObOr0Shoxplft1ZjAv8qPDg)
But before doing so, I want to make sure the FPKM was calculated from the total count of TE and refGene, right?
Many thanks for developing this awesome tool!

[emattei]

Hi @MaxwellShih,
I started using SQuiRE recently and I am facing all the problems mentioned in the issues which haven't been corrected yet.
I also would like to use TPM and I thin you should use the read_count field as input for the TPM calculation.
I think you can consider the read_count as the raw_counts.

[MaxwellShih]

Hello @emattei ,
Thanks for your comments. To calculate TPMs from raw counts, I have to get the gene length information. However, I found it's not easy to get the gene length information, at least for me. So, I just did it from FPKM.

[emattei]

I see, and in the denominator you used the sum of genes and TEs FPKM?