<p>As you surmised, kallisto requires a transcriptome fasta file, not a genome fasta file.</p>

CC BY-SA 4.0
2021-02-03T09:34:32.177Z
2021-02-03T09:34:32.177Z
Devon Ryan
<p>I am trying to run this kallisto (latest) quant and all I get are abundance and json files.</p>
<pre><code>kallisto quant --plaintext -t 4  -o rf-stranded.nob.1K1_L7.0 -i mm9_ref.idx  --rf-stranded 1K1_L7.0 2K1_L7.0
</code></pre>
<p>the results files in stderr is:</p>
<pre><code>[quant] fragment length distribution will be estimated from the data
[index] k-mer length: 15
[index] number of targets: 23
[index] number of k-mers: 363,704,628
[index] number of equivalence classes: 1,945,936
[quant] running in paired-end mode
[quant] will process pair 1: 1K1_L1.0
                             2K1_L1.0
[quant] finding pseudoalignments for the reads ... done
[quant] processed 22,897,424 reads, 4,476,549 reads pseudoaligned
[quant] estimated average fragment length: 24.2427
[   em] quantifying the abundances ... done
[   em] the Expectation-Maximization algorithm ran for 52 rounds
</code></pre>
<hr />
<p>I get an abundance file with:</p>
<pre><code>target_id       length  eff_length      est_counts      tpm
chr1    197195432       1.89745e+08     253015  21.3249
chr10   129993255       1.31621e+08     210320  25.5544
chr11   121843856       1.38776e+08     263606  30.3773
chr12   121257530       1.23195e+08     143568  18.6371
chr13   120284312       1.22484e+08     132969  17.3613
chr14   125194864       1.20926e+08     112711  14.9058
chr15   103494974       1.07866e+08     109335  16.2102
chr16   98319150        9.70075e+07     109045  17.9767
chr17   95272651        1.06375e+08     169166  25.4322
chr18   90772031        9.23542e+07     121990  21.1241
chr19   61342430        7.23377e+07     157142  34.7407
chr2    181748087       1.81791e+08     244870  21.5414
chr3    159599783       1.45978e+08     209257  22.9247
chr4    155630120       1.61978e+08     226893  22.4014
chr5    152537259       1.65672e+08     194951  18.8186
chr6    149517037       1.43243e+08     246652  27.5373
chr7    152524553       1.82795e+08     197527  17.2812
chr8    131738871       1.50556e+08     203954  21.6643
chr9    124076172       1.30446e+08     222639  27.295
chrX    166650296       1.40278e+08     176259  20.0942
chrY    15902555        5.76797e+07     123.462 0.0342311
mtDNA   16299   11804.8 737285  998819
rDNA    249368  720854  33272.5 738.159
</code></pre>
<p>run_info.json:</p>
<pre><code>        &quot;n_targets&quot;: 23,
        &quot;n_bootstraps&quot;: 100,
        &quot;n_processed&quot;: 22897424,
        &quot;n_pseudoaligned&quot;: 4476549,
        &quot;n_unique&quot;: 3641646,
        &quot;p_pseudoaligned&quot;: 19.6,
        &quot;p_unique&quot;: 15.9,
        &quot;kallisto_version&quot;: &quot;0.46.1&quot;,
        &quot;index_version&quot;: 10,
        &quot;start_time&quot;: &quot;&quot;,
        &quot;call&quot;: &quot;kallisto quant --plaintext -t 4 -b 100 -o rf-stranded.1K1_L1.0 -i mm9_ref.idx --bias --rf-stranded --gtf UCSC_genes.gtf --pseudobam --genomebam 1K1_L1.0.gz 2K1_L1.0.gz&quot;
}
</code></pre>
<hr />
<p>When I ran it with boot strapping (-b), -gtf and --pseudobam --genomebam it gave me a bam  file.</p>
<pre><code>44879990 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
8078785 + 0 mapped (18.00% : N/A)
44879990 + 0 paired in sequencing
22439995 + 0 read1
22439995 + 0 read2
8077160 + 0 properly paired (18.00% : N/A)
8077160 + 0 with itself and mate mapped
1625 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ&gt;=5)
</code></pre>
<p>I'm not sure what is going wrong, can someone help?</p>
<p>NOTE I use a 15nt kmer, and this about 1/2 of what is suggested (31 I think), is that a problem?</p>

CC BY-SA 4.0
2021-02-03T01:00:05.233Z
Thanks I think I know what is going on.  I probably should give a file of transcripts to the index step and not a chromosome file.  This is why it is giving my only chromosome abundances.
Modoc Dogman
CC BY-SA 4.0
2021-02-03T01:04:18.267Z
That makes sense. I would suggest posting that as an answer to your own question and then accepting it, to help others.
Maximilian Press
CC BY-SA 4.0
2021-02-03T01:05:05.490Z
OK but I first will try to verify.
Modoc Dogman
<rna-seq><kallisto>
Kallisto psudoaligner quant not giving me expected output
# Kallisto psudoaligner quant not giving me expected output

(Asked by: username_0 on 2021-02-02)

I am trying to run this kallisto (latest) quant and all I get are abundance and json files.
kallisto quant --plaintext -t 4  -o rf-stranded.nob.1K1_L7.0 -i mm9_ref.idx  --rf-stranded 1K1_L7.0 2K1_L7.0

the results files in stderr is:
[quant] fragment length distribution will be estimated from the data
[index] k-mer length: 15
[index] number of targets: 23
[index] number of k-mers: 363,704,628
[index] number of equivalence classes: 1,945,936
[quant] running in paired-end mode
[quant] will process pair 1: 1K1_L1.0
                             2K1_L1.0
[quant] finding pseudoalignments for the reads ... done
[quant] processed 22,897,424 reads, 4,476,549 reads pseudoaligned
[quant] estimated average fragment length: 24.2427
[   em] quantifying the abundances ... done
[   em] the Expectation-Maximization algorithm ran for 52 rounds


I get an abundance file with:
target_id       length  eff_length      est_counts      tpm
chr1    197195432       1.89745e+08     253015  21.3249
chr10   129993255       1.31621e+08     210320  25.5544
chr11   121843856       1.38776e+08     263606  30.3773
chr12   121257530       1.23195e+08     143568  18.6371
chr13   120284312       1.22484e+08     132969  17.3613
chr14   125194864       1.20926e+08     112711  14.9058
chr15   103494974       1.07866e+08     109335  16.2102
chr16   98319150        9.70075e+07     109045  17.9767
chr17   95272651        1.06375e+08     169166  25.4322
chr18   90772031        9.23542e+07     121990  21.1241
chr19   61342430        7.23377e+07     157142  34.7407
chr2    181748087       1.81791e+08     244870  21.5414
chr3    159599783       1.45978e+08     209257  22.9247
chr4    155630120       1.61978e+08     226893  22.4014
chr5    152537259       1.65672e+08     194951  18.8186
chr6    149517037       1.43243e+08     246652  27.5373
chr7    152524553       1.82795e+08     197527  17.2812
chr8    131738871       1.50556e+08     203954  21.6643
chr9    124076172       1.30446e+08     222639  27.295
chrX    166650296       1.40278e+08     176259  20.0942
chrY    15902555        5.76797e+07     123.462 0.0342311
mtDNA   16299   11804.8 737285  998819
rDNA    249368  720854  33272.5 738.159

run_info.json:
        "n_targets": 23,
        "n_bootstraps": 100,
        "n_processed": 22897424,
        "n_pseudoaligned": 4476549,
        "n_unique": 3641646,
        "p_pseudoaligned": 19.6,
        "p_unique": 15.9,
        "kallisto_version": "0.46.1",
        "index_version": 10,
        "start_time": "",
        "call": "kallisto quant --plaintext -t 4 -b 100 -o rf-stranded.1K1_L1.0 -i mm9_ref.idx --bias --rf-stranded --gtf UCSC_genes.gtf --pseudobam --genomebam 1K1_L1.0.gz 2K1_L1.0.gz"
}


When I ran it with boot strapping (-b), -gtf and --pseudobam --genomebam it gave me a bam  file.
44879990 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
8078785 + 0 mapped (18.00% : N/A)
44879990 + 0 paired in sequencing
22439995 + 0 read1
22439995 + 0 read2
8077160 + 0 properly paired (18.00% : N/A)
8077160 + 0 with itself and mate mapped
1625 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

I'm not sure what is going wrong, can someone help?
NOTE I use a 15nt kmer, and this about 1/2 of what is suggested (31 I think), is that a problem?

**Tags:** rna-seq, kallisto
> **Comments:**
> **username_0**: Thanks I think I know what is going on.  I probably should give a file of transcripts to the index step and not a chromosome file.  This is why it is giving my only chromosome abundances.  
> **username_1**: That makes sense. I would suggest posting that as an answer to your own question and then accepting it, to help others.  
> **username_0**: OK but I first will try to verify.  

---

## 1 answers

### Answer by username_2 on 2021-02-03. Score: 3

As you surmised, kallisto requires a transcriptome fasta file, not a genome fasta file.


---

