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TSV file for sample

Input files for Sarek can be specified using a tsv file given to the --sample parameter. The tsv file is a Tab Separated Value file with columns: subject gender status sample lane fastq1 fastq2 or subject gender status sample bam bai. The content of these columns should be quite straight-forward:

  • subject designate the subject, it should be the ID of the Patient, or if you don't have one, it could be the Normal ID Sample.
  • gender is the gender of the Patient, (XX or XY)
  • status is the status of the Patient, (0 for Normal or 1 for Tumor)
  • sample designate the Sample, it should be the ID of the Sample (it is possible to have more than one tumor sample for each patient)
  • lane is used when the sample is multiplexed on several lanes
  • fastq1 is the path to the first pair of the fastq file
  • fastq2 is the path to the second pair of the fastq file
  • bam is the bam file
  • bai is the index

All examples are given for a normal/tumor pair. If no tumors are listed in the TSV file, then the workflow will proceed as if it was a single normal sample instead of a normal/tumor pair.

Example TSV file for a normal/tumor pair with FASTQ files

In this sample for the normal case there are 3 read groups, and 2 for the tumor. It is recommended to add the absolute path of the paired FASTQ files, but relative path should work also. Note, the delimiter is the tab (\t) character:

G15511    XX    0    C09DFN    C09DF_1    pathToFiles/C09DFACXX111207.1_1.fastq.gz    pathToFiles/C09DFACXX111207.1_2.fastq.gz
G15511    XX    0    C09DFN    C09DF_2    pathToFiles/C09DFACXX111207.2_1.fastq.gz    pathToFiles/C09DFACXX111207.2_2.fastq.gz
G15511    XX    0    C09DFN    C09DF_3    pathToFiles/C09DFACXX111207.3_1.fastq.gz    pathToFiles/C09DFACXX111207.3_2.fastq.gz
G15511    XX    1    D0ENMT    D0ENM_1    pathToFiles/D0ENMACXX111207.1_1.fastq.gz    pathToFiles/D0ENMACXX111207.1_2.fastq.gz
G15511    XX    1    D0ENMT    D0ENM_2    pathToFiles/D0ENMACXX111207.2_1.fastq.gz    pathToFiles/D0ENMACXX111207.2_2.fastq.gz

Example TSV file for a normal/tumor pair with BAM files

On the other hand, if you have BAMs (T/N pairs that were not realigned together) and their indexes, you should use a structure like:

G15511    XX    0    C09DFN    pathToFiles/G15511.C09DFN.md.real.bam    pathToFiles/G15511.C09DFN.md.real.bai
G15511    XX    1    D0ENMT    pathToFiles/G15511.D0ENMT.md.real.bam pathToFiles/G15511.D0ENMT.md.real.bai

All the files will be created in the Preprocessing/NonRealigned/ directory, and by default a corresponding TSV file will also be deposited there. Generally, getting MuTect1 and Strelka calls on the preprocessed files should be done by:

nextflow run /shared/ucl/depts/cancer/apps/nextflow_pipelines/Sarek/main.nf -profile legion --sample Preprocessing/NonRealigned/mysample.tsv --step realign
nextflow run /shared/ucl/depts/cancer/apps/nextflow_pipelines/Sarek/somaticVC.nf -profile legion --sample Preprocessing/Recalibrated/mysample.tsv --tools Mutect2,Strelka

Example TSV file for a normal/tumor pair with recalibrated BAM files

The same way, if you have recalibrated BAMs (T/N pairs that were realigned together) and their indexes, you should use a structure like:

G15511    XX    0    C09DFN    pathToFiles/G15511.C09DFN.md.real.bam    pathToFiles/G15511.C09DFN.md.real.bai
G15511    XX    1    D0ENMT    pathToFiles/G15511.D0ENMT.md.real.bam    pathToFiles/G15511.D0ENMT.md.real.bai

All the files will be in he Preprocessing/Recalibrated/ directory, and by default a corresponding TSV file will also be deposited there. Generally, getting MuTect1 and Strelka calls on the recalibrated files should be done by:

nextflow run /shared/ucl/depts/cancer/apps/nextflow_pipelines/Sarek/somaticVC.nf -profile legion --sample Preprocessing/Recalibrated/mysample.tsv --tools Mutect2,Strelka

Input FASTQ file name best practices

The input folder, containing the FASTQ files for one individual (ID) should be organized into one subfolder for every sample. All fastq files for that sample should be collected here.

ID
+--sample1
+------sample1_lib_flowcell-index_lane_R1_1000.fastq.gz
+------sample1_lib_flowcell-index_lane_R2_1000.fastq.gz
+------sample1_lib_flowcell-index_lane_R1_1000.fastq.gz
+------sample1_lib_flowcell-index_lane_R2_1000.fastq.gz
+--sample2
+------sample2_lib_flowcell-index_lane_R1_1000.fastq.gz
+------sample2_lib_flowcell-index_lane_R2_1000.fastq.gz
+--sample3
+------sample3_lib_flowcell-index_lane_R1_1000.fastq.gz
+------sample3_lib_flowcell-index_lane_R2_1000.fastq.gz
+------sample3_lib_flowcell-index_lane_R1_1000.fastq.gz
+------sample3_lib_flowcell-index_lane_R2_1000.fastq.gz

Fastq filename structure:

  • sample_lib_flowcell-index_lane_R1_1000.fastq.gz and
  • sample_lib_flowcell-index_lane_R2_1000.fastq.gz

Where:

  • sample = sample id
  • lib = indentifier of libaray preparation
  • flowcell = identifyer of flow cell for the sequencing run
  • lane = identifier of the lane of the sequencing run

Read group information will be parsed from fastq file names according to this:

  • RGID = "sample_lib_flowcell_index_lane"
  • RGPL = "Illumina"
  • PU = sample
  • RGLB = lib