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HiSeq 2000

  1. Make sure sample sheet does not include quotes.
    • requires index sequence to only be six bases
  2. configureBclToFastq.pl --input-dir <BaseCalls_dir> --output-dir <Unaligned> --sample-sheet <BaseCalls_dir>/SampleSheet.csv --no-eamss --mismatches 1 --fastq-cluster-count 0
    • output-dir should be under fastq directory, year, run folder, then fastq.
  3. cd output-dir
  4. On mal: (screen or) nohup make -j 4
  5. concat read1, read2 fastq files
  6. Put symlinks in production folder pointing to fastq files
  7. Copy data from illumina-9 to rawseq folder
  8. Copy reports and status files to runReports folder, make link in run table

NextSeq 500

  1. Copy data from learfan back to rawseq directory.
  2. Copy sample sheet to top directory of run folder, name must be SampleSheet.csv
    • requires all 7 bases of index in sample sheet
  3. On mal: (screen or) nohup bcl2fastq2 --runfolder-dir ~/hlab/reorg/rawseq/<year>/<run folder> -p 3 -d 2 --barcode-mismatches 1
    • needs min 16G RAM per core (3x16=48G out of 60G)
  4. Make symlinks in fastq folder to rawseq Data/Intensities/BaseCalls
  5. Put symlinks in production folder pointing to fastq files


  1. rsync rawdata back to bx from makova-miseq-cache.
  2. Convert to fastq if necessary (similar to HiSeq)

HiSeq at Huck

  1. Get fastq files and Fastqc reports from ftp site (lab user name and password required).

Trouble shooting

  1. Things to make sure before you begin
  2. Make sure the file has unix line endings. If it was created using Excel on a Mac, it will have MacOSX line endings. Run Cathy’s line-ends program to change the line endings.
    • ~cathy/bin/line-ends
      Usage: /afs/bx.psu.edu/home/cathy/bin/line-ends <target> <filename> > output
      where target = win, mac, or unix
      Example: /afs/bx.psu.edu/home/cathy/bin/line-ends unix run26_SampleSheet.csv > run26_SampleSheet_endunix.csv

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