Kallisto: Estimated average fragment length
0
0
Entering edit mode
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
ADD COMMENT
0
Entering edit mode

Can you show us the exact command you used?

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

ADD REPLY
0
Entering edit mode

Attached more information ....

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] processed 48,339,659 reads, 29,727,059 reads pseudoaligned
[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
ADD REPLY
0
Entering edit mode

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?

ADD REPLY

Login before adding your answer.

Traffic: 2480 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6