Dear BioStars community,
I am using STARsolo to analyse the multiome protocol sc data, focusing exclusively on gene expression (GEX) at this stage.
Q1:
Since both R1 and R2 are 150 bp, I can use both reads for alignment, following the 10x chemistry SC5P-PE protocol. However, to ensure proper processing R1 that contains CB (16 bp) + UMI (12 bp) and needs to be clipped off using -- clip5pNbases 28 0
I also, can see Poly(T) sequences present in R1. For the aligner to use both R1 and R2 we need to have only cDNA in R1 left, how would you insure that polyT are also removed ? Can --clip5pNbases parameter insure that polyTs are also removed? I'd increase the length, but not sure to how much? -- clip5pNbases X 0
@A00690:382:HKCKTDMXY:2:1101:1723:1031 1:N:0:AAGATTGGAT+NAATCCCGCT
TACTGGCCATAGCTTGAACTGTCTTCATGTTTTTTTTTTTTTTTTTTTTTTTAAATTTAAACCCAGAACAATTAAAATATTGAACCATTTTTTAACAAGTTTCGGGAAGGGTTTTTAAAATATGATTGAACCAAAAAAAAAAAAGATAAG
+
F,F,FFFFFFFFFF:FFFF:FFF,F,:F,F:F:FFFFFFFFFFF:FFFFFFF,:,::F,:,F,,F,,,F::,:FFF,,:,,,,FF:::F,:FFF:,,:::F:,,,,,,,,,,:F:,FF,,,,:,,F,,,,,,,F:F:F:FFF,:,,FF,,
@A00690:382:HKCKTDMXY:2:1101:2953:1031 1:N:0:AAGATTGGAT+NAATCCCGCT
GGGTGTTGTCACAGCGGGAGTAATAAACTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTGTGAAAACAATAAAAAAAAAAAAATTTTAAAAAACATAAAATAAAAACAAAAACCAAAAACCAGGGTTATTTAGGTTTTTATTTTTAAAAA
+
FFFFFFFFFFFFFFFFFFFFFF,FFFFF,FFFFFF:FFFFFFFFFFF,FFFFFFFF,,:,,,FFF,,FF,FF:,,,,,::F,FF:,,FF,::,,,,,,,F,,FF,,,FFF,F:FFFF,:,,F:,:,F,,,F:,,F,F:F,,FFF,,FFF,
@A00690:382:HKCKTDMXY:2:1101:3007:1031 1:N:0:AAGATTGGAT+NAATCCCGCT
TTTCTCACATGCTCCCAATCCTGAGGGTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTGTATGTTTGAAATTTTTTTTTTGAAAATATTTAATATTTGAAAAATTAAAAAAAAAGAGGAAAATATAAAAAAAAAAACCAAAAAT
+
FFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFF:F:FFFFFFFF:FF:FFFFFFFFFFFFFFF:F,,,,,,:,,,:,,:F,,,,:F,:,F,,::,,:,,,,,:,,FF:::F,FFFF,,,,,,,,,,,,::,:,,F:F:::,,:,F,,FF,
Q2: For gene expression (GEX), my understanding is that the whitelist remains unchanged. However, for ATAC-seq, the barcodes should be reverse complementary as NovaSeq 6000 (v1.5) was used. Is that correct?
same question posted at STAR issues
thank you
