Problems with mapsplice

[rakscalloni]

Hi, guys!


I'm trying to align my reads using Mapsplice. However, I am having problems with bowtie index. I built my index using the file Homo_sapiens.GRCh38.dna.primary_assembly.fa, but I ran Mapsplice, it did not recognized the indexes.
Does anybody used Mapsplice? How do you build your indexes?

[mastal]

What happens if you use one of the pre-built bowtie indexes?

Does your reference have the same chromosome names as the index?

[GenoMax]

@rakscalloni: It generally helps us if you post the specific error message along with the command line you are using.

[rakscalloni]

Right!
Here is part of the output. The index files were downloaded from Bowtie web site.

[Wed Dec 2 11:51:51 2015] Beginning Mapsplice run (MapSplice v2.1.8)
[Wed Dec 2 11:51:51 2015] Bin directory: /dados/raks/MapSplice-v2.1.8/bin/
[Wed Dec 2 11:51:51 2015] Preparing output location /dados/raks/ESCs/mapsplice_esc/SRR1732022_mapsplice/
[Wed Dec 2 11:51:51 2015] Checking files or directory: /dados/raks/ESCs/mapsplice_esc/SRR1732022_1.fastq
[Wed Dec 2 11:51:51 2015] Checking files or directory: /dados/raks/ESCs/mapsplice_esc/SRR1732022_2.fastq
[Wed Dec 2 11:51:51 2015] Checking files or directory: /dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/
[Wed Dec 2 11:51:51 2015] Checking Bowtie index files
[Wed Dec 2 11:51:51 2015] Building Bowtie index for reference sequence
Could not open index file for writing: "dados/raks/ESC/mapsplice_esc/Homo_sapiens.GRCh38.3.ebwt"
Please make sure the directory exists and that permissions allow writing by
Bowtie.
Command: /dados/raks/MapSplice-v2.1.8/bin/bowtie-build /dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/10.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/1.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens
.GRCh38/11.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/12.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/13.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/14.fa,/dados/raks/ESCs/mapsplice
_esc/Homo_sapiens.GRCh38/15.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/16.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/17.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/18.fa,/dados/ra
ks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/19.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/2.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/20.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38
/21.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/22.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/3.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/4.fa,/dados/raks/ESCs/mapsplice_esc/Homo
_sapiens.GRCh38/5.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/6.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/7.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/8.fa,/dados/raks/ESCs/mapsp
lice_esc/Homo_sapiens.GRCh38/9.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/X.fa,/dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/Y.fa dados/raks/ESC/mapsplice_esc/Homo_sapiens.GRCh38
[MapSplice Running Failed]
Error: Splice sequence indexing failed

[mastal]

I don't understand what you are trying to do.

If you have downloaded pre-built Bowtie indexes, then you don't need to build the index again with MapSplice.

Is the output you have shown above, the output you get when you run 'bowtie-build'?

Have you looked at the MapSplice user guide?

http://www.netlab.uky.edu/p/bioinfo/MapSpliceManual#input

[mastal]

OK, I think I get what is happening, correct me if I am wrong.

You are trying to align the reads using MapSplice (it would help if you had posted the command you used), MapSplice can't find the bowtie indexes, so it tries to build the indexes,
but this also fails, and the program comes to a halt.

Two things to check: Did you put the pre-built indexes in a directory where MapSplice expects to find them, and are the permissions for that directory set so that MapSplice can read the indexes?

From the output above, it looks like maybe the '/' is missing at the beginning of the path to the index files.

[rakscalloni]

Actually, Mapsplice finds the pre-built indexes, but it can't read the file "Homo_sapiens.GRCh38.3.ebwt"

The indexes are in the correct directory and I don't think it is a permission problem, because is only one of 6 index files that Mapsplice is not recognizing...

My command line

python /dados/raks/MapSplice-v2.1.8/mapsplice.py -p 1 -o /dados/raks/ESCs/mapsplice_esc/SRR1732018_mapsplice --qual-scale phred33 --bam --min-fusion-distance 200 --fusion-non-canonical --gene-gtf /dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38.79.gtf -c /dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38 -x dados/raks/ESC/mapsplice_esc/Homo_sapiens.GRCh38 -1 /dados/raks/ESCs/mapsplice_esc/SRR1732018_1.fastq -2 /dados/raks/ESCs/mapsplice_esc/SRR1732018_2.fastq

[rakscalloni]

Also, when I remove the option "-x" and let Mapsplice build the indexes, the analysis did not work and the output files are empty.

[mastal]

-x dados/raks/ESC/mapsplice_esc/Homo_sapiens.GRCh38 -1 /dados/raks/ESCs/mapsplice_esc/SRR1732018_1.fastq -2 /dados/raks/ESCs/mapsplice_esc/SRR1732018_2.fastq

Looks like the path to your index files has 2 problems:
1. not starting the path with '/'
2. 'ESC' instead of 'ESCs'

By the way, the Bowtie1 and Bowtie2 indexes are not compatible. The MapSplice2 manual says it uses the Bowtie1 indexes, so check that those were the indexes you downloaded.

[rakscalloni]

I correct the "ESC" error and checked the indexes. The "/" has to be used because of the server I am using... I ran Mapsplice again and got the message above. And I have no idea about how fix this problem...

[Thu Dec 3 09:35:14 2015] Beginning Mapsplice run (MapSplice v2.1.8)
[Thu Dec 3 09:35:14 2015] Bin directory: /dados/raks/MapSplice-v2.1.8/bin/
[Thu Dec 3 09:35:14 2015] Preparing output location /dados/raks/ESCs/mapsplice_esc/SRR1732019_mapsplice/
[Thu Dec 3 09:35:14 2015] Checking files or directory: /dados/raks/ESCs/mapsplice_esc/SRR1732019_1.fastq
[Thu Dec 3 09:35:14 2015] Checking files or directory: /dados/raks/ESCs/mapsplice_esc/SRR1732019_2.fastq
[Thu Dec 3 09:35:14 2015] Checking files or directory: /dados/raks/ESCs/mapsplice_esc/Homo_sapiens.GRCh38/
[Thu Dec 3 09:35:14 2015] Checking Bowtie index files
[Thu Dec 3 09:35:14 2015] Inspecting Bowtie index files
[Thu Dec 3 09:35:15 2015] Checking reference sequence length
[Thu Dec 3 09:35:45 2015] Checking consistency of Bowtie index and reference sequence
Error: Reference name in Bowtie Index contains space:
'chr1 AC:CM000663.2 gi:568336023 LN:248956422 rl:Chromosome M5:6aef897c3d6ff0c78aff06ac189178dd AS:GRCh38' contains space
[MapSplice Running Failed]
Error: Checking consistency of Bowtie index and reference sequence failed
Please check if Bowtie Index and Reference Sequence parameters are set correctly and they comply with MapSplice requirements
Visit MapSplice 2.0 online manual for details:

http://www.netlab.uky.edu/p/bioinfo/MapSplice2UserGuide#CommandLine

[mastal]

Where did you get the reference genome from?

You have to check two things:
1. That the chromosome names in the reference genome fasta headers are in the same format as in the bowtie index.

2. that the fasta headers are in the format used by MapSplice.
See the link in your error message above. MapSplice seems to be objecting to spaces in tthe header lines.

[GenoMax]

@Mastal has already pointed out things you need to address.

I suggest that you just make new bowtie1 indexes using the fasta reference you have on hand since your indexes do not match the reference. Make sure there are no spaces in fasta headers (replace them with an "_" instead).