CountPeaks error when using Singleron Biotechnologies 3'-end BAM files

[hkarakurt8742]

Hello everyone, I am using Sierra with a Singleron Biotechnologies based scRNA-Seq data. From my previous post, it is said that I can use Singleron datasets. I found peaks without and error but when I use CountPeaks function, I have an error as:

There are 2054 whitelist barcodes. Import genomic features from the file as a GRanges object ... OK Prepare the 'metadata' data frame ... OK Make the TxDb object ... OK There are 35175 sites Doing counting for each site... Error in (function (classes, fdef, mtable) : unable to find an inherited method for function 'writeMM' for signature '"NULL"' In addition: Warning message: In .get_cds_IDX(mcols0$type, mcols0$phase) : The "phase" metadata column contains non-NA values for features of type stop_codon. This information was ignored.

And my barcodes looks like this: "AAAAAACCGTTA AAAACAGAAGAG AAAACCGCACTT AAAACTGCGTCC AAAAGCTTCAGA AAAAGTTTCCGC AAAATAGCTAAC AAACACTTAGGG AAACAGGTTTGA AAACCCAATAAC AAACCTCTCACG AAACGCCAATCA AAACGGCATCAG AAACTCCCTTAT AAACTTCGCAAG AAACTTGGAACT AAAGAAACCCAT AAAGCATACCGT AAAGCATACTTT AAAGCCTGCTTG AAAGGCCGATCT AAAGGCGGGCAG"

Normally barcodes have "-1" as I know but they do not. Can it be the problem? What might be the problem?

Thank you in advance.

[rj-patrick]

Hi @hkarakurt8742, Can you check what format your cell barcodes are in with your bam file? i.e. can you show:

view your_bam.bam | head -n 1

Cheers, Ralph

[hkarakurt8742]

Hello @rj-patrick. Thank you for your reply.

The output of "samtools view Sample.out.sorted.bam | head -n 1"

AGAGCCCTCCTC_TTGACACC_9153775 16 1 14559 255 148M * 0 0 TTGAAGCTGGTCTCCACACAGGGCTGGTTCCGTCACCCCCTCCCAAGGAAGTAGGTCTGAGCAGCTTGTCCTGGCTGTGTCCATGTCAGAGCAACGGCCCAAGTCTGGGTCTGGGGGGGAAGGTGTCATGGAGCCCCCTACGATTCCC <7JF7JA7JA-<-JJAJJJFA)JFJJFFFJFJAF-JJJJF<JJJJJJJJJJFJJF-JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJF7FAA NH:i:1 HI:i:1 AS:i:144 nM:i:1

[rj-patrick]

Hi @hkarakurt8742,

The issue is you BAM is missing the cell barcode (CB:Z) that Sierra requires (as well as the UMI tag UB:Z). I'm guessing the cell barcode information is included in the header, but you will need to assign it to a tag for Sierra to recognise it. For reference, one of the bam files included with the Vignette is formatted as follows (note the CB:Z and UB:Z tags):

• 0 1 74044733 255 14M242491N83M1S 0 0 * CB:Z:TCACAAGGTACCGAGA-1 UB:Z:AATAAATGAT

Hope that helps.

[hkarakurt8742]

Hi @hkarakurt8742,

The issue is you BAM is missing the cell barcode (CB:Z) that Sierra requires (as well as the UMI tag UB:Z). I'm guessing the cell barcode information is included in the header, but you will need to assign it to a tag for Sierra to recognise it. For reference, one of the bam files included with the Vignette is formatted as follows (note the CB:Z and UB:Z tags):

• 0 1 74044733 255 14M242491N83M1S 0 0 * CB:Z:TCACAAGGTACCGAGA-1 UB:Z:AATAAATGAT

Hope that helps.

Hello @rj-patrick, Thank you for your reply. I found the problem and I think this information can be useful for Singleron platform users. The CeleScope tool that developed by Singleron Biotechnologies produces several outputs in different folders. For Sierra, users can use "sample_aligned_posSorted_addTag.bam" that located in "02.featureCounts" folder. Other bam files developed by this tool does not involve barcodes.

Thank you again.