additional sequences

[ljy-sys]

Thanks for this tools to help us analysis the smartseq3 data，but when i using the parameter “additional_files” to add a exogenous sequence，the result without this sequence is not a umicount of 0， other expression of other genes was consistent with that without this sequence. so, I sincerely hope to get your help, thank you.

this is my .yaml file:

project: Q2Q4
sequence_files:
file1:
name: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/0-merged_fq/Q2Q4.merged.R1.fastq.gz
base_definition:
- cDNA(23-150)
- UMI(12-19)
find_pattern: ATTGCGCAATG
file2:
name: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/0-merged_fq/Q2Q4.merged.R2.fastq.gz
base_definition:
- cDNA(1-150)
file3:
name: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/0-merged_fq/Q2Q4.merged.I1.fastq.gz
base_definition:
- BC(1-8)
file4:
name: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/0-merged_fq/Q2Q4.merged.I2.fastq.gz
base_definition:
- BC(1-8)
reference:
STAR_index: /storage1/project_raw/auto/test_with_CAR/reference/reference/star
GTF_file: /storage1/project_raw/auto/test_with_CAR/reference/genes.gtf
additional_STAR_params: '--limitSjdbInsertNsj 2000000 --clip3pAdapterSeq CTGTCTCTTATACACATCT'
additional_files: /storage1/project_raw/auto/test_with_CAR/reference/CAR.fa
out_dir: /storage1/project_raw/auto/test_with_CAR/zUMIs/smartseq3/Q2Q4
num_threads: 32
mem_limit: 100
filter_cutoffs:
BC_filter:
num_bases: 3
phred: 20
UMI_filter:
num_bases: 3
phred: 20
barcodes:
barcode_num: 96
barcode_file: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/barcode.txt
automatic: no
BarcodeBinning: 1
nReadsperCell: 100
counting_opts:
introns: yes
downsampling: '0'
strand: 0
Ham_Dist: 1
velocyto: no
primaryHit: yes
twoPass: no
make_stats: yes
which_Stage: Filtering
Rscript_exec: Rscript
STAR_exec: STAR
pigz_exec: pigz
samtools_exec: samtools

[ljy-sys]

Thanks for this tools to help us analysis the smartseq3 data，but when i using the parameter “additional_files” to add a exogenous sequence，the result without this sequence is not a umicount of 0， other expression of other genes was consistent with that without this sequence. so, I sincerely hope to get your help, thank you.

this is my .yaml file:

project: Q2Q4 sequence_files: file1: name: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/0-merged_fq/Q2Q4.merged.R1.fastq.gz base_definition: - cDNA(23-150) - UMI(12-19) find_pattern: ATTGCGCAATG file2: name: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/0-merged_fq/Q2Q4.merged.R2.fastq.gz base_definition: - cDNA(1-150) file3: name: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/0-merged_fq/Q2Q4.merged.I1.fastq.gz base_definition: - BC(1-8) file4: name: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/0-merged_fq/Q2Q4.merged.I2.fastq.gz base_definition: - BC(1-8) reference: STAR_index: /storage1/project_raw/auto/test_with_CAR/reference/reference/star GTF_file: /storage1/project_raw/auto/test_with_CAR/reference/genes.gtf additional_STAR_params: '--limitSjdbInsertNsj 2000000 --clip3pAdapterSeq CTGTCTCTTATACACATCT' additional_files: /storage1/project_raw/auto/test_with_CAR/reference/CAR.fa out_dir: /storage1/project_raw/auto/test_with_CAR/zUMIs/smartseq3/Q2Q4 num_threads: 32 mem_limit: 100 filter_cutoffs: BC_filter: num_bases: 3 phred: 20 UMI_filter: num_bases: 3 phred: 20 barcodes: barcode_num: 96 barcode_file: /storage1/project_raw/auto/test_with_CAR/smartseq3/Q2Q4/barcode.txt automatic: no BarcodeBinning: 1 nReadsperCell: 100 counting_opts: introns: yes downsampling: '0' strand: 0 Ham_Dist: 1 velocyto: no primaryHit: yes twoPass: no make_stats: yes which_Stage: Filtering Rscript_exec: Rscript STAR_exec: STAR pigz_exec: pigz samtools_exec: samtools

and i got the file "additional_sequence_annot.gtf", and the geges.gtf contain the additional information.

[cziegenhain]

Hi,

Sorry I do not understand the issue, what do you mean with "the result without this sequence is not a umicount of 0"?

[ljy-sys]

Hi,

Sorry I do not understand the issue, what do you mean with "the result without this sequence is not a umicount of 0"?

En, this means that the inserted sequence does not appear in umicount statistics, and the gene_names.txt file also does not contain the gene.

[cziegenhain]

Could you confirm that the BAM file has any aligned reads for the added sequence from the fasta file?
You could eg. run samtools idxstat on the .filtered.Aligned.GeneTagged.sorted.bam.bai for this.