Question: Kallisto, --genomebam, and IGV visualization problem
0
gravatar for jtoker
5 months ago by
jtoker0
jtoker0 wrote:

I am currently trying to use kallisto and IGV to visualize reads from a single-cell RNASeq experiment. I'm interested in non-coding RNA, but I would still like to see how the reads align to each chromosome. Currently, I can visualize how the reads align to each individual non-coding transcript by aligning to the ensembl non-coding RNA fasta. But when I tried implementing --genomebam and the GTF file from ensembl, I stopped seeing my reads when I aligned to the genome fasta. My code below successfully generates my transcript index:

! kallisto index -i transcripts.idx Homo_sapiens.GRCh38.ncrna.fa.gz

But then I try the below code to generate a bam file to use in IGV, such that I see how the reads align to the genome...

! kallisto quant --genomebam --gtf Homo_sapiens.GRCh38.99.gtf.gz -i transcripts.idx -o output 1.fastq 2.fastq

I get a bam file successfully, but I don't see any alignments in IGV.

For what it's worth, I get the below warning:

Warning: 173489 transcripts were defined in GTF file, but not in the index

I've read in the kallisto manual that I may also need a chromosome number/size file, but I don't know how to get such a file.

Thanks so much

rna-seq bam kallisto igv alignment • 354 views
ADD COMMENTlink written 5 months ago by jtoker0
1

You can get the chromosome number/size file from UCSC. GRCH38 example.

Are you mixing and matching reference genome/transcriptome from two places (e.g. Ensembl/UCSC)?

ADD REPLYlink modified 5 months ago • written 5 months ago by genomax89k

No, my reference genome and transcriptome are both from ensembl's website. I'll try incorporating the chromosome file.

Thanks

ADD REPLYlink written 5 months ago by jtoker0
1

UCSC uses chr notation in front of chromosome numbers but Ensembl does not. So if you use that file make sure ID's match by removing chr.

ADD REPLYlink written 5 months ago by genomax89k
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