Open ttn1883 opened 2 years ago
Hi @ttn1883
For the standard 10X 3' libraries, please use parameters as described here: https://github.com/alexdobin/STAR/blob/master/docs/STARsolo.md
Cheers Alex
Hi @alexdobin
I followed the exact same guide but my alignment was 4%. For the 3' protocol, CB is 16 and UMI is 12. and the dTs is 30.
Therefore, I used --clip5pNbaes 58 2 and --soloBarcodeMate 2 to specify that mate2 is the one with the CB/UMI.
Thank you
Hi @ttn1883
with standard parameters, you need to use --readFilesIn Read2 Read1
, i.e. the first file should be cDNA read and the 2nd file should be the barcode read.
Hi @alexdobin
I did that as well. Thank for your time to troubleshoot. Please see my complete codes below:
STAR --genomeLoad NoSharedMemory --outSAMattributes All --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts --readFilesCommand zcat --runThreadN 16 --sjdbGTFfile /GCF_016700215.2_bGalGal1.pat.whiteleghornlayer.GRCg7w_genomic.gtf --outReadsUnmapped Fastx --outMultimapperOrder Random --genomeDir /STAR_indices/ --readFilesIn /R2_001.fastq.gz /R1_001.fastq.gz --outFileNamePrefix /Taylor_012 --soloCBwhitelist /3M-february-2018.txt --soloType CB_UMI_Simple --soloBarcodeMate 2 --clip5pNbases 28 2 --soloCBstart 1 --soloCBlen 16 --soloUMIstart 17 --soloUMIlen 12
Hi @ttn1883
in the latest parameters set, please remove --soloBarcodeMate 2
.
Also, I am not sure why you are using --clip5pNbases 28 2
I removed --soloBarcodeMate 2 and --clip5pNbases 28 2 and received the following error. Will try adding --soloBarcodeReadLength 0 next.
STAR --genomeLoad NoSharedMemory --outSAMattributes All --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts --readFilesCommand zcat --runThreadN 16 --sjdbGTFfile /GCF_016700215.2_bGalGal1.pat.whiteleghornlayer.GRCg7w_genomic.gtf --outReadsUnmapped Fastx --outMultimapperOrder Random --genomeDir /STAR_indices/ --readFilesIn /R2_001.fastq.gz /R1_001.fastq.gz --outFileNamePrefix /Taylor_012 --soloCBwhitelist /3M-february-2018.txt --soloType CB_UMI_Simple --soloCBstart 1 --soloCBlen 16 --soloUMIstart 17 --soloUMIlen 12
STAR version: 2.7.10a compiled: 2022-01-14T18:50:00-05:00 :/home/dobin/data/STAR/STARcode/STAR.master/source
Aug 31 12:09:02 ..... started STAR run Aug 31 12:09:03 ..... loading genome Aug 31 12:09:06 ..... processing annotations GTF Aug 31 12:09:13 ..... inserting junctions into the genome indices Aug 31 12:09:31 ..... started mapping
EXITING because of FATAL ERROR in input read file: the total length of barcode sequence is 150 not equal to expected 28 Read ID=@A00738:420:HY77NDSX3:3:1101:1217:1000 ; Sequence=NGCTCCATCCATCTCGGTCTTCAATTGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGAGCTGTAAAAACCCCGGATGCAGGCACCCAAAATACCCACCAATAAAAACAAATTAAATAATTTAAAATTGACACATAAAAATTAAAGAA SOLUTION: check the formatting of input read files. If UMI+CB length is not equal to the barcode read length, specify barcode read length with --soloBarcodeReadLength To avoid checking of barcode read length, specify --soloBarcodeReadLength 0 Aug 31 12:09:31 ...... FATAL ERROR, exiting
Removing -soloBarcodeMate 2 and --clip5pNbases 28 2 and then adding --soloBarcodeReadLength 0 works. However, it is essentially the same as --soloBarcodeLength 150. Meaning that we are completely ignoring the cDNA is Read1. So far no solution allows using both Read1 and Read2 cDNA for mapping.
My libraries were build using the 10X Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1.
My Read 1 sequence looks like this: @A00738:420:HY77NDSX3:3:1101:1217:1000 1:N:0:CGGAACCCAA+TCCTCGAATC NGCTCCATCCATCTCGGTCTTCAATTGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGAGCTGTAAAAACCCCGGATGCAGGCACCCAAAATACCCACCAATAAAAACAAATTAAATAATTTAAAATTGACACATAAAAATTAAAGAA
When I used --soloBarcodeReadLength 150. I got 68.4% but basically, I am ignoring the data from Read 1.
When I tried: --soloType CB_UMI_Simple --soloBarcodeMate 2 --clip5pNbases 58 2 --soloCBstart 1 --soloCBlen 16 --soloUMIstart 17 --soloUMIlen 12, alignment went down from 68% to 4%.
I input my Read 2 first and Read 1 second. Read 1 includes the CB/UMI/Adapter/cDNA sequences.
Attached is the log.out output.
Thank you for your input.