Kallisto: Estimated average fragment length
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8 weeks ago

I used kallisto tool for RNA-seq quantification of pair-end reads. At the end of the quantification, it was reported by the tool that the estimated average fragment length is zero. When I look at the output abundance file, I have quantification TPM values which are not zero. So, I hope there is no problem with alignment. what do I have the average fragment length as zero?

RNA-Seq alignment next-gen sequencing • 195 views
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Can you show us the exact command you used?

And can you show us a few lines of the output abundance file?

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Command:

kallisto quant -t 4 -i kallisto_index -o ./ SRR6250323_1.fastq.gz SRR6250323_2.fastq.gz SRR6250324_1.fastq.gz SRR6250324_2.fastq.gz


Output:

[quant] fragment length distribution will be estimated from the data
[index] k-mer length: 31
[index] number of targets: 109,480
[index] number of k-mers: 97,929,191
[index] number of equivalence classes: 469,538
[quant] running in paired-end mode
[quant] will process pair 1: SRR6250323_1.fastq.gz
SRR6250323_2.fastq.gz
[quant] will process pair 2: SRR6250324_1.fastq.gz
SRR6250324_2.fastq.gz
[quant] finding pseudoalignments for the reads ... done
[quant] estimated average fragment length: 0
[   em] quantifying the abundances ... done
[   em] the Expectation-Maximization algorithm ran for 1,477 rounds

Abundance file: (only few lines)
target_id   length  eff_length  est_counts  tpm
ENSDART00000152292  2584    2585    0   0
ENSDART00000185434  1761    1762    0   0
rna34937    1983    1984    0   0
ENSDART00000078734  4537    4538    0   0
ENSDART00000157481  4535    4536    0   0
TCONS_00124789  1657    1658    0   0
TCONS_00124790  1617    1618    355 10.6338
TCONS_00102373  7439    7440    39.7846 0.259169
TCONS_00102374  7397    7398    1.91578 0.0125508

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Thanks, something is indeed off.

The eff_length should generally be the length minus the average fragment length + 1. Here, the eff_length is the only the length + 1. This will affect your results.

A few suggestions: Make sure you're using the latest version of kallisto (0.46.0). Additionally, make sure you're building the index corrrectly (and that you have downloaded the correct index / references).

Can you tell me what index / reference you're using?