STARsolo 2.7.0e - A problem in reading the barcode file

[Miri]

Hi all

I'm trying to use the latest version (2.7.0e) of STARsolo, but for some reason, it can't read the barcode file.

This is the error I get:

EXITING because of FATAL ERROR in input read file: the total length of barcode sequence is 0 not equal to expected 26

Read ID=@A00187:37:HFM7VDMXX:2:1101:1687:1000 1 N 0   Sequence=

SOLUTION: make sure that the barcode read is the second in --readFilesIn and check that is has the correct formatting

If UMI+CB length is not equal to the barcode read length, specify barcode read length with --soloBarcodeReadLength

And this is the head of the file:

@A00187:37:HFM7VDMXX:1:1101:1723:1000 1:N:0:TCAGCCGT

TTCGGTCAGCTAACAATACATCCTTG

+

FFF:FFFFFFFFFFFF:FFFFFFF:F

@A00187:37:HFM7VDMXX:1:1101:1958:1000 1:N:0:TCAGCCGT

GACGTGCGTGAGGGTTCCATAGGTCC

+

FFFFFFFFFFFFFFFFFFFFFFFFFF

@A00187:37:HFM7VDMXX:1:1101:2446:1000 1:N:0:TCAGCCGT

TTGACTTAGAGTCTGGACTACAGTTC

+

FFFFFFFFFFFFFFFFFFFFFFFFFF

Any suggestion can help here.

Thanks!

Miri.

[Alexander Dobin]

Hi Miri,

please make sure that the barcode file is 2nd in --readFilesIn, i.e.

--readFilesIn cDNAfragment.fastq.gz Barcode.fastq.gz

Note that for 10X protocol this means that Read2 goes first, i.e.

--readFilesIn Read2.fq.gz Read1.fq.gz

Cheers

Alex

[Miri]

Now I realize that I put a comma between the file names instead of a space. 

Now it's running.

Thanks for the support and your excellent aligner! 

Miri. 

[Kathie Mihindukulasuriya]

Hi Alex,

I am trying to run STARsolo (2.7.1a) but it does not recognize my 10X barcode file:

 /home/mihinduk/STAR-2.7.1a/bin/Linux_x86_64/STAR --runThreadN 10 --runMode alignReads --soloType Droplet --soloCBwhitelist /usr/local/genome/cellranger-3.0.2/cellranger-cs/3.0.2/lib/python/cellranger/barcodes/3M-february-2018.txt.gz  --readFilesIn TWEK-blood_pool-blood_pool-lib1_S5_L003_R2_001.fastq.gz TWEK-blood_pool-blood_pool-lib1_S5_L003_R1_001.fastq.gz --readFilesCommand zcat --soloFeatures GeneFull --genomeDir /home/public/kathie/GRCh38-3.0.0.premrna --outFileNamePrefix TWEK-blood_ --outStd BAM_SortedByCoordinate --outReadsUnmapped Fastx

EXITING because of FATAL ERROR in input CB whitelist file: /usr/local/genome/cellranger-3.0.2/cellranger-cs/3.0.2/lib/python/cellranger/barcodes/3M-february-2018.txt.gz the total length of barcode sequence is 85 not equal to expected 26

SOLUTION: make sure that the barcode read is the second in --readFilesIn and check that is has the correct formatting

gzip -cd /usr/local/genome/cellranger-3.0.2/cellranger-cs/3.0.2/lib/python/cellranger/barcodes/3M-february-2018.txt.gz | head

AAACCCAAGAAACACT

AAACCCAAGAAACCAT

AAACCCAAGAAACCCA

AAACCCAAGAAACCCG

AAACCCAAGAAACCTG

AAACCCAAGAAACGAA

AAACCCAAGAAACGTC

AAACCCAAGAAACTAC

AAACCCAAGAAACTCA

AAACCCAAGAAACTGC

gzip -cd TWEK-blood_pool-blood_pool-lib1_S5_L003_R1_001.fastq.gz | head

@A00580:81:HKLV3DSXX:3:1101:2329:1016 1:N:0:TTGTTGAT

TCAATCTAGTAACCTCGCCCTATCTCGC

+

FFFFFFFFFFFFFFFFFFFFFFFFFFFF

@A00580:81:HKLV3DSXX:3:1101:2383:1016 1:N:0:TTGTTGAT

GTGGAGAAGAAGGGATGTTTATATTGAG

+

FFFFFFFFFFFFFFFFFFFFFFFFFFFF

@A00580:81:HKLV3DSXX:3:1101:2672:1016 1:N:0:TTGTTGAT

CCTCCTCCAGACTCTATATGGGATTCGC

I tried adding --soloBarcodeReadLength 85

But then I get this:

EXITING because of FATAL ERROR in input CB whitelist file: /usr/local/genome/cellranger-3.0.2/cellranger-cs/3.0.2/lib/python/cellranger/barcodes/3M-february-2018.txt.gz the total length of barcode sequence is 85 not equal to expected 85

SOLUTION: make sure that the barcode read is the second in --readFilesIn and check that is has the correct formatting

Help!

Kathie

[Alexander Dobin]

Hi Kathie,

please unzip the whitelist file 3M-february-2018.txt.gz, it has to be plain text file.

Cheers

Alex

[Kathie Mihindukulasuriya]

That fixed the problem.  Thanks

[Kathie Mihindukulasuriya]

Hi Alex,

We are trying STARsolo for single nuclei RNAseq.   We have been using a premRNA reference and GTF with CellRanger.  Is STARsolo creating this under the hood from the genomic reference and GTF or should we be using the premRNA reference, like we  did with CellRanger?

Thank you for your help,

Kathie

[Alexander Dobin]

Hi Kathie,

you can run STARsolo with --soloFeatures GeneFull option, and it will generate the gene-cell count matrix for premRNA, i.e. consider overlap between the reads and full gene (exons+introns).

You can also run --soloFeatures Gene GeneFull, which will create both the normal (mature RNA) exonic count and pre-mRNA count.

Cheers

Alex

[Jonathan Keats]

Hi Alex,

Is there a special setting or way you need to create the genome index to support GeneFull.  I'm getting a segmentation fault with any --soloFeatures option including "GeneFull" where:

Gene

Gene SJ

work fine while:

GeneFull

Gene GeneFull

Gene SJ GeneFull

all create a segmentation fault after first stage mapping (tested 2.7.1a and 2.7.2a)

Aug 28 12:32:14 ..... started STAR run
Aug 28 12:32:14 ..... loading genome
Aug 28 12:32:42 ..... started 1st pass mapping
Segmentation fault (core dumped)

Example Code block

STAR \
    --runMode alignReads \
    --runThreadN 19 \
    --genomeDir /home/tgenref/homo_sapiens/grch38_hg38/hg38tgen/gene_model/ensembl_v97/tool_resources/star_2.7.1a/100bpReads \
    --genomeLoad NoSharedMemory \
    --twopassMode Basic \
    --sjdbOverhang 99 \
    --readFilesType Fastx \
    --readFilesIn \
    MC1685_0003_3_PB_MNC_C1_X5SCR_F01501_G3C84_AACCCGTT_L001_R2_001.fastq.gz \
    MC1685_0003_3_PB_MNC_C1_X5SCR_F01501_G3C84_AACCCGTT_L001_R1_001.fastq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix MC1685_0003_3_PB_MNC_C1_X5SCR_ \
    --outSAMtype BAM SortedByCoordinate \
    --outSAMmode Full \
    --outSAMunmapped None \
    --outSAMmapqUnique 255 \
    --outSAMattributes NH HI AS nM CR CY UR UY \
    --soloType Droplet \
    --soloCBwhitelist /packages/cellranger/3.0.2/cellranger-cs/3.0.2/lib/python/cellranger/barcodes/737K-august-2016.txt \
    --soloFeatures Gene SJ GeneFull \
    --soloUMIdedup 1MM_All \
    --soloCBstart 1 \
    --soloCBlen 16 \
    --soloUMIstart 17 \
    --soloUMIlen 10 \
    --soloBarcodeReadLength 0 \
    --soloStrand Reverse \
    --soloOutFileNames Solo.out/ MC1685_0003_3_PB_MNC_C1_X5SCR_genes.tsv MC1685_0003_3_PB_MNC_C1_X5SCR_barcodes.tsv MC1685_0003_3_PB_MNC_C1_X5SCR_matrix.mtx MC1685_0003_3_PB_MNC_C1_X5SCR_matrixSJ.mtx MC1685_0003_3_PB_MNC_C1_X5SCR_matrixGeneFull.mtx

Maybe its an issue with the ensembl v97 GTF used to build the index?

[Alexander Dobin]

Hi Jonathan,

please try the later releases 2.7.1a or 2.7.2a as some bugs have been fixed.

Or even the development branch https://github.com/alexdobin/STAR/tree/soloDevelop which will soon be released.

If it still causes seg-faults, please send me the Log.out file.

Cheers

Alex

[Bryce Turner]

Hi Alex,

I've attached the Log.out for the example mentioned by Jonathan's earlier post. Let me know if any other information is needed!

Thank you for your help,

Bryce

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[Alexander Dobin]

Hi Bryce,

thanks - nothing suspicious in the Log.out file...

Could you please try to run it with --readMapNumber 1000 (or higher until it seg-faults), and then cut that many reads from the fastq R1 and R2, and send them to me?

Thanks!

Alex

[Alexander Dobin]

Hi Bryce,

thanks for the files!

I think the problem is related to the 2-pass mode, and it was fixed on the development branch:

https://github.com/alexdobin/STAR/tree/soloDevelop

Please try it out and let me know if the problem is fixed. I will make a 2.7.3 release out of it next week, hopefully.

Cheers

Alex