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Velocyto: Not found cell and umi barcode in entry of the bam file

I'm trying to get the fraction of spliced and unspliced genes to after calculate the RNA velocity with velocyto.

When I run this command:

velocyto run -u Gene -o ./Data_RNAv ./data1.bam ./GenomeIndex/gencodev38annotation.gtf

I get the following Error message:

 The bam file does not contain cell and umi barcodes appropriatelly formatted.

This is my workflow so far:

  1. Downloaded the two fastq files using the sratoolkit

  2. Downloaded hg38.fa and the reference .gtf file

  3. Created the genome index using STAR

Like this:

STAR --runMode genomeGenerate  --genomeDir ./GenomeIndex --genomeFastaFiles ./GenomeInde /hg38.fa --sjdbGTFfile ./GenomeIndex/gencodev38annotation.gtf
  1. Aligned the genome using STAR

Like this:

STAR --runThreadN 24 --genomeDir ./GenomeIndex --sjdbGTFfile ./GenomeIndex/gencodev38annotation.gtf --sjdbOverhang 100 --outSAMtype BAM Unsorted --readFilesIn ./data_Day4/SRR9127057_S1_L001_R1_001.fastq ./H9_D4/SRR9127057_S1_L001_R2_001.fastq
  1. Using velocyto.py to writing out a standard loom file: and here is where I get the error saying that the UMI is not found in the bam file

What did I do wrong?

velocyto scrna-seq umi rna
• 3,219 views • link
written by bs58 • bs58

Probably the SRA data does not have UMI/Barcode sequence in the header. You can check that information in the fastq header.

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written by Nitin Narwade Nitin Narwade

This is the begining of the fastq file that I have

>head ./data_Day4/SRR9127057_S1_L001_R1_001.fastq
@SRR9127057.1 A00291:31:H5W5MDMXX:2:1101:1579:1000 length=25
TGTTACCCNGCTCGTCGTTATGCCG
+SRR9117954.1 A00291:31:H5W5MDMXX:2:1101:1579:1000 length=25
,#FFFFFFF:FF:FFFFFFFFFFFF
@SRR9127057.2 A00291:31:H5W5MDMXX:2:1101:2338:1000 length=25
NGCTGTCCAAGGAAGCTAGTCCACT
+SRR9117954.2 A00291:31:H5W5MDMXX:2:1101:2338:1000 length=25
F#FFFFFFFFFFFFFFFF:FFFFFF
@SRR9127057.3 A00291:31:H5W5MDMXX:2:1101:3007:1000 length=25
TNAACTTTGCGTGGTCTCCTCAAGC

How can I know if it has the UMI/Barcode?

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written by bs58 • bs58

1 answer

From the description, this was a 10xGenomics single cell dataset. The cell barcode and UMI information is in read 2, but STAR doesn't understand that. Either use STARSolo, or cellranger.

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written by swbarnes2 swbarnes2

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