Kallisto did not generate layers using rna velocity tutorial

[230101-valentina]

Dear all, I am running Kallisto in python to get spliced and unspliced attributes for RNA-velocity. I used the tutorial for Constructing a velocity index with kb (https://www.kallistobus.tools/tutorials/kb_velocity_index/python/kb_velocity_index.html) for human genome, as explained.

!wget ftp://ftp.ensembl.org/pub/release-104/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz !wget ftp://ftp.ensembl.org/pub/release-104/gtf/homo_sapiens/Homo_sapiens.GRCh38.104.gtf.gz

!pip install kb-python==0.25.0

!kb ref -i index.idx -g t2g.txt -f1 cdna.fa -f2 intron.fa -c1 cdna_t2c.txt -c2 intron_t2c.txt --workflow lamanno -n 8 \ Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz \ Homo_sapiens.GRCh38.104.gtf.gz

it works properly and than I use !kb count to built h5ad count matrix in this way:

!kb count --h5ad -i index.idx_cdna -g t2g.txt -x 10xv2 -o IPS_RNAvelocity_matrix \ -c1 cdna_t2c.txt -c2 intron_t2c.txt --filter bustools -t 2 \ 2_IPS_S2_L001_R1_001.fastq.gz \ 2_IPS_S2_L001_R2_001.fastq.gz \ 2_IPS_S2_L002_R1_001.fastq.gz \ 2_IPS_S2_L002_R2_001.fastq.gz

it works but when I opened the adata file it has no layers:

adata AnnData object with n_obs × n_vars = 54999 × 60664 var: 'gene_name'

Can u help me with this issue? Thanks

[Lioscro]

Hi, @230101-valentina, I think there is a missing --workflow lamanno in your count command. Could you try running this instead?

!kb count --h5ad -i index.idx_cdna -g t2g.txt -x 10xv2 -o IPS_RNAvelocity_matrix \
-c1 cdna_t2c.txt -c2 intron_t2c.txt --filter bustools -t 2 --workflow lamanno \
2_IPS_S2_L001_R1_001.fastq.gz \
2_IPS_S2_L001_R2_001.fastq.gz \
2_IPS_S2_L002_R1_001.fastq.gz \
2_IPS_S2_L002_R2_001.fastq.gz \

[230101-valentina]

Thank you, I solved the problem as you said and get the correct matrix with layers. I was wondering to know if there is a possibility to get the human t2g.txt index including also gene symbol. Thank you

Inviato da Postahttps://go.microsoft.com/fwlink/?LinkId=550986 per Windows

Da: Joseph @.> Inviato: mercoledì 15 settembre 2021 22:35 A: @.> Cc: Murtaj @.>; @.> Oggetto: Re: [pachterlab/kb_python] Kallisto did not generate layers using rna velocity tutorial (#145)

Hi, @230101-valentinahttps://github.com/230101-valentina, I think there is a missing --workflow lamanno in your count command. Could you try running this instead?

!kb count --h5ad -i index.idx_cdna -g t2g.txt -x 10xv2 -o IPS_RNAvelocity_matrix \

-c1 cdna_t2c.txt -c2 intron_t2c.txt --filter bustools -t 2 --workflow lamanno \

2_IPS_S2_L001_R1_001.fastq.gz \

2_IPS_S2_L001_R2_001.fastq.gz \

2_IPS_S2_L002_R1_001.fastq.gz \

2_IPS_S2_L002_R2_001.fastq.gz \

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[230101-valentina]

Dear, I have another question, as I found in the release page (https://github.com/pachterlab/kb_python/releases) that kb version 0.25.0 = "When the matrix is converted to H5AD or Loom format (using the --h5ad or --loom options), the gene/feature names are included as a column in the var of the anndata." This did not work with the RNAvelocity count matrix building, as the t2g.txt file did not contain the third column with gene name, right? How can I get the gene name too? If I build by myself a custom RNAvelocity index for human, with this version I would be able to get also gene names? and to visualized them in the analysis using scanpy? Thank you very much

[Lioscro]

Hi, @230101-valentina, I believe the t2g should contain the gene names. Are you using a version older than 0.25.0?

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