SC5P-R2 sequencing

[yarloz-old]

Hello!

How should I describe bc:umi:seq structure in -x parameter for 5' 10x protocol where both reads are used for alignment (SC5P-R2 - https://kb.10xgenomics.com/hc/en-us/articles/115003764132-How-does-cellranger-count-auto-detect-chemistry-)? Following variants were tried - 0,0,16:0,16,26:0,26,0 (coding sequence in the 1st read), 0,0,16:0,16,26:1,0,0 (coding sequence in the 2nd read) but in both cases we are losing information from the other read.

Best regards,
Yaroslav Lozinsy

[chrarnold]

I would also like to know how to specify this. Had / Have the same problem for an SC5P-R2 chemistry, and the issue closing without a comment did not help me...

[yarloz-old]

Option for processing of both reads was added to the last versions of kallisto. I do not remember exactly how to get this information so just try to check out the code or kallisto updates notes for different versions. пт, 20 нояб. 2020 г. в 23:32, chrarnold <[email protected]>:

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I would also like to know how to specify this. Had / Have the same problem for an SC5P-R2 chemistry, and the issue closing without a comment did not help me... — You are receiving this because you authored the thread. Reply to this email directly, view it on GitHub <#226 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/AJXJQJSPSO62QLJD74G4RC3SQ3G7PANCNFSM4IPPE3WQ> .

-- С уважением, Лозинский Ярослав (Lozinsky Yaroslav)

[chrarnold]

Thanks for the quick reply! I used the latest version as part of KITE, but failed to make kallisto bus work correctly. Barcodes were just not detected with kallisto despite being present. I played around with the chemistry string, similar to your description, but with no luck unfortunately so far.

[kikegoni]

Hey,

First, thanks a lot for implementing the Kallisto-Bustools, it is super helpful! I was wondering hoy this should be done. I have scRNA-seq which are from 5' 10x protocol and they have 150 bp (both in R1 and R2). R1 has the first 16bp as the CB, then the next 10bp are the UMI and then there is the cDNA, whereas R2 contains only the cDNA.

In your release notes I have found that this situation has been adressed with kallisto 0.46.2 https://github.com/pachterlab/kallisto/releases

I have tried this

kallisto bus -i $KALLISTO_INDEX -o $OUTPUT_DIR -x 0,0,16:0,16,26:0,26,0:1,0,0 -t 8 $FILE1 $FILE2

Throwing the following error:
Error: technology string must contain two colons (:), three found: "0,0,16:0,16,26:0,26,0:1,0,0"
Unable to create technology: 0,0,16:0,16,26:0,26,0:1,0,0

How should I indicate to the seq triplet that it contains the cDNA in part of R1 and in the entire R2?

Thanks a lot for any help you might provide about this and sorry for the inconveniences.

Best,

Kike