Question about RiboTools Kmer analysis - phasing

[jjb91]

Hello,

I aligned my adaptor-trimmed and rRNA-removed reads to sacCer3 genome using Bowtie. I ran the Kmer software using sorted Bam files and the correct GFF3 file downloaded from Ensemble. The Kmer analysis returns an HTML file containing data. It shows a graph displaying the number of reads for each Kmer size. However, the phasing graphs for individual sizes is displaying null values. I was wondering if I am doing anything incorrect or if this is a new issue.

Thank you in advance.

[jamesp]

Hello,
we think this issue is because you used a fasta file (from UCSC) and a GFF3 file (from Ensembl) from different sources.
We suggest you also use the corresponding fasta file for your dataset from Ensembl. It looks like kmer while its able to count the number of reads cannot match the gene names to the GFF3 file.
Unfortunately this is a bit of rework to obtain the sorted bam file.
Thanks to let us know if this works.

Kind Regards
jamesp

[jjb91]

(24-Apr-2017 02:27 PM)jamesp Wrote:  Hello,
we think this issue is because you used a fasta file (from UCSC) and a GFF3 file (from Ensembl) from different sources.
We suggest you also use the corresponding fasta file for your dataset from Ensembl. It looks like kmer while its able to count the number of reads cannot match the gene names to the GFF3 file.
Unfortunately this is a bit of rework to obtain the sorted bam file.
Thanks to let us know if this works.

Kind Regards
jamesp

Thank you, James. I redid the alignment with the fasta file I obtained form Ensemble. Now when I do the Kmer analysis, I receive an error like the one below.

Quote:Error during footprinting : list index out of range
Programme aborted at Mon Apr 24 18:35:14 2017

Begin kmer and phasing analysis at Mon Apr 24 18:34:55 2017
2295780 reads are presents in your bam file

[jamespa]

Dear jjb,
there are over 350 datasets in your history and so without spending a lot of time its difficult for us to follow what you did. Please let us know the names and history id of the datasets in chronological order which were involved in all the different steps leading up to this error.
for example:
bowtie alignment ????????
bam sort ??????
kmer 348 Kmer report on data 272 and data 344

You can send this to RiboGalaxy@gmail.com

or alternatively you could use copy datasets (from the icon at the top right of your history ) to copy these datasets into a new history. Then it should be easier for us to understand exactly what you did and we can investigate further.

Kind Regards
jamesp

[jjb91]

(24-Apr-2017 08:12 PM)jamespa Wrote:  Dear jjb,
there are over 350 datasets in your history and so without spending a lot of time its difficult for us to follow what you did. Please let us know the names and history id of the datasets in chronological order which were involved in all the different steps leading up to this error.
for example:
bowtie alignment ????????
bam sort ??????
kmer 348 Kmer report on data 272 and data 344

You can send this to RiboGalaxy@gmail.com

or alternatively you could use copy datasets (from the icon at the top right of your history ) to copy these datasets into a new history. Then it should be easier for us to understand exactly what you did and we can investigate further.

Kind Regards
jamesp

Sorry for the confusion, James.

I have copied a sample list of relevant data sets into their own history and sent them to the provided email. I truly appreciate the help!

[jamesp]

This issue was finally resolved by using a gff and fasta file for the alignments from NCBI
https://www.ncbi.nlm.nih.gov/genome/
The files are usually found by clicking on the appropriate links at the top of the page after you search for your species.
For the future, we would recommend using files from NCBI for analysis using Ribo Tools.
Regards
Jamesp