CAGEscan

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Introduction

CAGEscan is a technology where paired-ends of random-primed 5'-capped molecules are sequenced, thus associating TSS to collections of downstream exonic sequences scanning the transcript. For a more complete description of CAGEscan and its potential application, please refer to:

Linking promoters to functional transcripts in small samples with nanoCAGE and CAGEscan. Nat Methods. 2010 Jul;7(7):528-34. Plessy C, Bertin N, Takahashi H, Simone R, Salimullah M, Lassmann T, Vitezic M, Severin J, Olivarius S, Lazarevic D, Hornig N, Orlando V, Bell I, Gao H, Dumais J, Kapranov P, Wang H, Davis CA, Gingeras TR, Kawai J, Daub CO, Hayashizaki Y, Gustincich S, Carninci P. (PDF)

FANTOM5 CAGEscan libraries were be produced from biological samples selected for the diversity of transcripts they produced.

The primary purpose of those libraries is to associate HelicosCAGE derived TSS (in particular orphan DPI clusters) to annotations such as known transcripts, and in last ressort to provide a partial reconstitution of the intron/exon structure of the TSS product.

Protocols

Datasets and links

  • LS-archive IDs: (search for "FANTOM5 CAGEScan") 841 (pilot) 861~866 (runs), 961~966 (re-sequenced lanes).
  • Library IDs: NCig10013-NCig10068, NCig10126-NCig10131
ls /analysisdata/mirrors/F5_file_current/UPDATE_024/f5pipeline/*/*NCig*.5prime.fq.gz | grep -o NCig..... | sort -u
  • DDBJ Sequence Read Archive, DRA005606 (2017).

First version of the alignments (until UPDATE_025 incl.)

Not used in the Data Descriptor.

(old links follow)

Re-aligned version for the data descriptor

  • Data was realigned with OP-WORKFLOW-CAGEscan-FANTOM5-v1.0, a derivative of the 2014 pipeline, with hardcoded information to ease processing of the FANTOM5 libraries with Grid Engine. [[1]]. Main reasons for realigning were a) G-correction and b) reproducible research.
  • Reads and alignments: [[2]].

Meta CAGEscan assembly

The score indicates the number of libraries contributing to the cluster. 

time cat */CAGEscan_clusters/*.bed |
/home/plessy/src/CAGEscan-Clustering/CAGEscan-Clustering.pl \
  --cluster_file /osc-fs_home/scratch/moirai/plessy/input/hg19.cage_peak_phase1and2combined_anncoord.bed \
  --bin_intersectbed $(which intersectBed) \
  --format bed > CAGEscan_metaclusters.bed

real	5m27.640s
user	6m23.873s
sys	0m2.293s

zenbu_upload -url http://fantom.gsc.riken.jp/zenbu -file CAGEscan_metaclusters.bed -assembly hg19 -name "CAGEscan metaclusters" -desc "FANTOM5 CAGEscan metaclusters"

Zenbu upload of individual libraries

Downloaded all the BED12 files from UPDATE_026 in a single directory.

URL decoding, modified from: http://stackoverflow.com/a/10660730/5309786 

# Returns a string in which the sequences with percent (%) signs followed by
# two hex digits have been replaced with literal characters.
rawurldecode() {

  # This is perhaps a risky gambit, but since all escape characters must be
  # encoded, we can replace %NN with \xNN and pass the lot to printf -b, which
  # will decode hex for us

  printf -v REPLY '%b' "${1//%/\\x}"
  printf "${REPLY}"
}

for file in *pairs.bed.gz
do
  printf "%s\t%s\t%s\n" \
    $file \
    $(echo $file | grep -o NCig10...) \
    "$(rawurldecode ${file%%.hg19.GCTATA.pairs.bed.gz} | sed 's/\./ /g') upload2016-12-26"
done > CAGEscan_pairs.txt

CAGEscan_clust

for file in *clusters.bed.gz
do
  printf "%s\t%s\t%s\n" \
    $file \
    $(echo $file | grep -o NCig10...) \
    "$(rawurldecode ${file%%.hg19.GCTATA.pairs.bed.gz} | sed 's/\./ /g') upload2016-12-26"
done > CAGEscan_clusters.txt

zenbu_upload -url http://fantom.gsc.riken.jp/zenbu -assembly hg19 -filelist CAGEscan_pairs.txt -singletag_exp
zenbu_upload -url http://fantom.gsc.riken.jp/zenbu -assembly hg19 -filelist CAGEscan_clusters.txt

Available as tracks in view http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=ZkJi4RdBAFhnsudxePrZxD (public collaboration "FANTOM5 CAGEscan").

Potential loci of interest

cat CAGEscan_metaclusters.bed |
  awk '{OFS="\t"} $6 == "+" {print $1, $2, $8, $4, $5, $6}' |
  bedtools subtract -s -a - -b /osc-fs_home/scratch/gmtu/annotation/homo_sapiens/gencode-18/gencode.v18.annotation.genes.bed |
  sort -k5,5n |
  grep p@chr

Pipelines and qsub scripts

for lib in $(ls /analysisdata/mirrors/F5_file_current/UPDATE_024/f5pipeline/*/*NCig*.5prime.fq.gz | grep -o NCig..... | sort -u )
do
  mkdir -p $lib
  pushd $lib
  sed "s/\[library_id]/$lib/g" ../../CAGEscan_short-reads.xml > $lib.xml
  printf 'MOIRAI_HOME=/home/scratch/moirai/bin\n'     >  $lib.sh
  printf 'DATA_HOME=/home/plessy/pipelines/%s\n' $lib >> $lib.sh
  printf '$MOIRAI_HOME/MoiraiExecute.pl $DATA_HOME/%s.xml\n' $lib >> $lib.sh
  printf '$MOIRAI_HOME/java -classpath $MOIRAI_HOME/Moirai.jar MoiraiUtility log  $DATA_HOME/%s.xml\n' $lib >> $lib.sh
  printf '$MOIRAI_HOME/java -classpath $MOIRAI_HOME/Moirai.jar MoiraiUtility html $DATA_HOME/%s.xml\n' $lib >> $lib.sh
  popd
done
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