BD Rhapsody single cell RNA: usable sequence

Question

[Darked89]

Hello,

I got 2x101bp fastq data obtained using BD Rhapsody single cell kit. Not sure if I am missing something, but with labeling in both r1 and r2 reads I am struggling to find usable human mRNA sequence.

In read_1 I got +70bp labels (polyT track vary in length apparently):

GCCTTACAAactggcctgcgaAGATAGTTCggtagcggtgacaACACTCCGCCTCCCGCCGGCACTGTTTGTTTTTTTTTTTTTCCTGGACAACCCATCTG

Linker1 and Linger2 small letters.

In read_2 the labels can get up to 95bp:

label with sequence tag 10
GTTGTCAAGATGCTACCGTTCAGAGGCAGCCGGCGTCGTACGAGGCACAGCGGAGACTAGATGAGGCCCCAAAAAAAAAAAAAAAAAAAAAAAAA

example sequence
GTTGTCAAGATGCTACCGTTCAGAGGCAGCCGGCGTCGTACGAGGCACAGCGGAGACTAGATGCGGCCCCAAAAAAAAAAAAAAAAAAAAAAGTTCCCGGT

Frankly I doubt there is much to map to human transcriptome, but I would be happy proven wrong.

My questions:

1. Is this a normal outcome of BDs Rhapsody single cell RNA protocol or there was some "one to many" labeling? I am thinking here about 95bp long labels in read_2 just to distinguish between samples.
2. Assuming such IMHO over the top labeling is the standard way of preparing the sample, would switching to say 2x150bp make the data way better?

My idea would be to skip the labels in read_2 and use a simple 8bp say Illumina index2, but I suspect that decoupling cell & molecule labeling in read_1 performed downstream from sequence tag labeling ending up in read_2 may not be easy.

Many thanks for your help

DK

edit

Looks like it was extra labeling:

The picture is from page 11 of https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/BD_Single_Cell_Genomics_Bioinformatics_Handbook.pdf

[colindaven]

You could check the mapping rate of the R2 reads only by alignment using STAR etc. These should be the mRNA reads. AFAIK they should not have any technical labels etc as per your diagram.

[Darked89]

There is a S3 bucket with stand alone BD Rhapsody scripts: http://bd-rhapsody-public.s3-website-us-east-1.amazonaws.com/Rhapsody-Install-Bundle/

Answer 1

[GenoMax]

Please see: BD Rhapsody software - single cell seq

You probably need to use their software for initial processing.

[Darked89]

Thank you. Makes sense, since scripting cut and paste of bunch of cell/molecule tags with possible substitutions is non-trivial.

Running it at "home" may be tricky: """ Whole Transcriptome Analysis (WTA) assays: 96 GB (>192 GB recommended) """

Not easy to find 96GB RAM machine running Docker. Maybe I can get around it using Singularity:

https://github.com/common-workflow-language/cwltool#using-singularity

[GenoMax]

I believe BD offers this via SevenBridges if that is a possibility.