Elementolab/Creating Custom SureSelect Libraries

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  • Download gene annotation from UCSC
wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/knownGene.txt.gz
wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/ensGene.txt.gz
wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene.txt.gz

unzip all .gz

  • Extract intervals corresponding to all exons and promoters in the region of interest (exons are extended by 100bp on either side)
perl ~/PERL_SCRIPTS/FindxGeneExonsInRegion.pl --chr=chr2 --start=11445451 --end=36017005 \
 --genefiles=knownGene.txt,ensGene.txt,ensGene.txt | perl /Users/olivier/PERL_SCRIPTS/merge_intervals_slow.pl - \
 > ETM2_intervals.txt
  • Calculate combined length
perl ~/PERL_SCRIPTS/count_sum_intervals.pl ETM2_intervals.txt
  • Estimate approximate coverage
perl ~/PERL_SCRIPTS/calculate_coverage.pl --mr=30 --readlen=51 --genomesize=2138647 --capture=0.7
  • Convert into eArray format
perl ~/PERL_SCRIPTS/interval2eArray.pl ETM2_intervals.txt > ETM2_intervals_eArray.txt 

  • Upload into eArray to create bait library. Important: you will need create two bait libraries, 1 with repeats off, 1 with repeats on.

Tile coverage: 2X default, try to get to 3X Overlap with repeat/avoid regions: default is 20bp, can set to 30bp.

  • Wait until upload of two libraries is processed, then download library designs, and unzip
  • Find baits that were not included because of repeats
perl ~/PERL_SCRIPTS/SureSelect_findBaitsInDesignNotInOther.pl ETM2_noRM/BaitTiling_tdt ETM2/BaitTiling_tdt > ETM2/repeat_baits.txt
  • convert to fasta format
perl ~/PERL_SCRIPTS/SureSelect_baits2fa.pl ETM2/repeat_baits.txt  > ETM2/repeat_baits.fa 
  • BLAT them against the human genome
blat hg19.fa ETM2/repeat_baits.fa ETM2/repeat_baits.fa.hg19blat.psl
  • retrieve baits and get statistics
perl ~/PERL_SCRIPTS/SureSelect_analyzeBlat.pl ETM2/repeat_baits.fa.hg19blat.psl ETM2_noRM/BaitTiling_tdt > ETM2/rescued_baits.txt
  • in eArray, create a new bait group/library using "Create a Library by Uploading Baits"
  • in eArray, create a new library using "Create Library from Existing Bait Group(s)". Select the repeat masked baits and the rescued baits.
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