Transcriptional and post-transcriptional regulation of gene expression

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Transcriptional and post-transcriptional
regulation of gene expression
protein
Translation Localization Stability
mRNA
3UTR
Pol II
DNA
Activation Repression
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• Where does each transcription factor bind in the
  genome, in each cell type, at a given time ? Near
  which genes ?
• What is the cis-regulatory code of each factor ?
  Does they require any co-factors ?

DNA
Activation Repression
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ChIP-seq
Transcription factor of interest
Antibody
Genome Analyzer II (Solexa)
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Control input DNA
Genome Analyzer II (Solexa)
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ACCAATAACCGAGGCTCATGCTAAGGCGTTAGCCACAGATGGAAGTCCGA
CGGCTTGATCCAGAATGGTGTGTGGATTGCCTTGGAACTGATTAGTGAAT
TC
TGGTTATTGGCTCCGAGTACGATTCCGCAATCGGTGTCTACCTTCAGGCT
GCCGAACTAGGTCTTACCACACACCTAACGGAACCTTGACTAATCACTTA
AG
Average length 250bp
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25-40bp
ACCAATAACCGAGGCTCATGCTAAGGCGTTAGCCACAGATGGAAGTCCGA
CGGCTTGATCCAGAATGGTGTGTGGATTGCCTTGGAACTGATTAGTGAAT
TC
TGGTTATTGGCTCCGAGTACGATTCCGCAATCGGTGTCTACCTTCAGGCT
GCCGAACTAGGTCTTACCACACACCTAACGGAACCTTGACTAATCACTTA
AG
Average length 250bp
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25-40bp
ACCAATAACCGAGGCTCATGCTAAGGCGTTAGCCACAGATGGAAGTCCGA
CGGCTTGATCCAGAATGGTGTGTGGATTGCCTTGGAACTGATTAGTGAAT
TC
TGGTTATTGGCTCCGAGTACGATTCCGCAATCGGTGTCTACCTTCAGGCT
GCCGAACTAGGTCTTACCACACACCTAACGGAACCTTGACTAATCACTTA
AG
Average length 250bp
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BCL6 ChIP-seq

• Lymphoma cell line (OCI-Ly1)
• Solexa/Illumina
• 6 lanes for ChIP, 1 for input DNA, 1 for QC
• 36nt long sequences
• 32 Million reads
• Aligned/mapped to hg18 with Eland

Melnick lab at WCMC
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Read mapping with Eland
Solexa Read
AAAATACGCGTATTCTCCCAAAACAATATC
AAAAATTCTCCCAAAACAAAAAAATACGCGTATTCTCCCAAAACAATATC
TTACAAGATGTAAATATACCCAAGATG
Reference Human Genome (hg18)
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Read mapping with Eland
Solexa Read
AAAATACGCCTATTCTCCCAAAACAATATC
AAAAATTCTCCCAAAACAAAAAAATACGCGTATTCTCCCAAAACAATATC
TTACAAGATGTAAATATACCCAAGATG
Reference Human Genome (hg18)
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Read mapping with Eland
Solexa Read
AAAATACGCCTATTCTCCCATAACAATATC
AAAAATTCTCCCAAAACAAAAAAATACGCGTATTCTCCCAAAACAATATC
TTACAAGATGTAAATATACCCAAGATG
Reference Human Genome (hg18)
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Reads can map to multiple locations/chromosomes
Solexa Read 1
Solexa Read 2
Reference Human Genome (hg18)
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Reads map to one strand or the other
Solexa Read 1
Solexa Read 2
hg18
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gtHWI-EAS83_30UCEAAXX129151011 AGGTCACAAAACAAGT
CCTAACAAATTTAAGAGTAT U0 1 13 62 chr8.fa 59699745 R
DD gtHWI-EAS83_30UCEAAXX128261245 GTCAGAAAAATC
CTTTTTATTATATAAACAATACAT U2 0 0 1 chr5.fa 12119509
8 F DD 15G 20G gtHWI-EAS83_30UCEAAXX12900945 G
TCATCAAACTCCAAGGATTCTGTTTTCAACATACT U0 1 1 0 chr18
.fa 8914049 R DD gtHWI-EAS83_30UCEAAXX121037111
8 GAAAGTGATTAGCAGATTGTCATTTAATAATTGTCT U2 0 0 1 ch
r1.fa 97496963 F DD 18G 28G gtHWI-EAS83_30UCEAAXX1
2898874 GATAAATTTTTTCCTACAATCTTAAATTATTACACA U
1 0 1 0 chr3.fa 95643444 R DD 10C gtHWI-EAS83_30UCE
AAXX12918928 AAAAATTAAACAATTCTAAAAATATTTTTATC
TTAA U2 0 0 1 chr2.fa 177727639 R DD 18C 31G gtHWI-
EAS83_30UCEAAXX1213244 GCACATGTCATACTCTTTCTAG
CTCTCTTATTTTTC U0 1 0 0 chr8.fa 79132719 R DD gtHWI
-EAS83_30UCEAAXX128991015 AAATTAATGTAAAAAATAGG
ATACTGAATTGTGATA U1 0 1 0 chr10.fa 69774166 F DD 3
0G gtHWI-EAS83_30UCEAAXX12909926 GTAGTTAACAATA
ATTTATTTTATACTTCAAAATTC U1 0 1 17 chrX.fa 26496842
R DD 7A gtHWI-EAS83_30UCEAAXX127011702 GTCAGAA
TTAATTAATCAAAACACCAAATGTACTTC U0 1 0 0 chr12.fa 72
700465 F DD gtHWI-EAS83_30UCEAAXX129961003 ATTT
TGACTTTATTATTTTTTCTTCAATGTTTTTAA NM 0 0 0 gtHWI-EAS
83_30UCEAAXX128841090 GAAAGTACATCAAATACATATTAT
ATACTTTACATA R2 0 0 2 gtHWI-EAS83_30UCEAAXX12911
937 AATCCATATACATTTCTTTTTAATCATTTCCTCTTT U1 0 1
0 chr11.fa 94204222 F DD 20G gtHWI-EAS83_30UCEAAXX
121517330 GTGAGTTTCTTAATCCTGAGTTCTAATTTTATTTCA
R0 29 255 255 gtHWI-EAS83_30UCEAAXX129041031 AC
ATTTTATAAATTTTTAATTTCATTTTAATTTATA NM 0 0 0 gtHWI-E
AS83_30UCEAAXX1212911469 GTTTTTAAAATCAACACTTTT
ATTATAGAAGTAGCA U0 1 0 1 chr12.fa 62166701 R DD gtH
WI-EAS83_30UCEAAXX121697828 GTACTGATGTAAACTTGG
TAAAAACATTGACATAAA U0 1 0 0 chr14.fa 65160857 F DD
gtHWI-EAS83_30UCEAAXX121415583 GAAGAAAATGACTAT
GTCAAAATATTATCTCTCAAT U0 1 0 0 chr5.fa 97782464 F
DD gtHWI-EAS83_30UCEAAXX1215611653 GTTTTACTGATT
TTCTTACTTACTAAACTACCTGTT U0 1 0 0 chr7.fa 13320026
5 F DD gtHWI-EAS83_30UCEAAXX121579943 AATGATACG
GCGACCACCGACAGGTTCAGAGTTCTA NM 0 0 0 gtHWI-EAS83_30
UCEAAXX121705268 GAGAATTATTCAGAAGTCAAATCTGTGCT
TAGTTTA U2 0 0 1 chr5.fa 162472124 R DD 3G 7C gtHWI
-EAS83_30UCEAAXX121489318 GTATGTATCATATATATTTA
TGTATCATATATATTT R1 0 3 2 gtHWI-EAS83_30UCEAAXX12
10031113 GATTGCTCCATTATTTGTTAAAAACATAGTAAAATA NM
0 0 0 gtHWI-EAS83_30UCEAAXX128951072 ATGAGATCA
GTACTTCAAAGAGATATCTGCACTCCC U0 1 1 9 chr12.fa 3383
0898 R DD gtHWI-EAS83_30UCEAAXX128531178 GTTAGT
CCCAATATTCCATTAATCCCAATAAATATA U2 0 0 1 chr6.fa 11
0722427 F DD 15G 19G gtHWI-EAS83_30UCEAAXX121432
972 GAGATAATAATAGCAGTTATGGCATCGAGATAATTT U0 1 0 0
chr2.fa 47305609 R DD gtHWI-EAS83_30UCEAAXX1217
18341 GTAGAGGGCACACATCACAAACAAGTTTCTGAGAAT R2 0 0
3 gtHWI-EAS83_30UCEAAXX121171302 GAATATCCACTTG
CAGACTTTACAAACAAATTTTTT R2 0 0 4 gtHWI-EAS83_30UCEA
AXX1210551126 GGCAGATGAAACTTCTATACACTATATTTTAG
CCAG U0 1 0 0 chr13.fa 90021137 F DD gtHWI-EAS83_30
UCEAAXX129711371 GAAAGAAAAACTATTGAAAAAATAGTTAC
TTTCCAA U0 1 0 0 chr1.fa 74303257 R DD gtHWI-EAS83_
30UCEAAXX121774614 GTGTAGATGATATCGAGGGCATTAGAA
GTAAATAGC U0 1 0 0 chr5.fa 16031200 F DD gtHWI-EAS8
3_30UCEAAXX121207808 GAGAGGAAATAATAAAGATAAAAGT
AGAAAAAGTGA U0 1 0 0 chr1.fa 187326417 F DD gtHWI-E
AS83_30UCEAAXX121680815 GATAATTATGTTGTTGTAATTA
TTGTTTGTTTTTTT U0 1 0 0 chr15.fa 46739015 R DD gtHW
I-EAS83_30UCEAAXX121688260 GTTGACAATCCAGCTGTCA
TAGAAACTGACTATTTT U0 1 0 0 chr12.fa 38910133 R DD
gtHWI-EAS83_30UCEAAXX121051916 AAAAATTCTCCCAAAA
CAACAAGATGTAAATATACC U0 1 0 0 chr3.fa 101625712 R
DD gtHWI-EAS83_30UCEAAXX121771308 GTTCTTACACTGA
TATGAAGAAATACCTGAGACTGG U0 1 2 67 chr2.fa 21412853
7 R DD gtHWI-EAS83_30UCEAAXX12911917 GAGAAACAC
ACATATTTTTGTAAGTGCCATCACATC U1 0 1 0 chr7.fa 13668
652 R DD 18C gtHWI-EAS83_30UCEAAXX121105348 GTA
TTATCTAACACACAAGATGATGTTTGTTTTTAT NM 0 0 0 gtHWI-EA
S83_30UCEAAXX121048857 GAGTGTAGAAAATTTTCTGCCCT
AAAATATTTGTTA U1 0 1 0 chr6.fa 74625385 F DD 13G gt
HWI-EAS83_30UCEAAXX127431729 GTATCCTAAAGTGTATC
TTATGTTTTTTCATCTTCT U1 0 1 0 chr12.fa 7400023 R DD
9C gtHWI-EAS83_30UCEAAXX12128764 AATAAAACAAAT
TCCAATGGCTTAGATTCTACTTAA U2 0 0 1 chr10.fa 9802079
9 R DD 15C 20C gtHWI-EAS83_30UCEAAXX129401059 A
AATGGTCATACTTCCCAAAGCGATCTACAGATTCA U1 0 1 29 chr3
.fa 50834510 R DD 19C gtHWI-EAS83_30UCEAAXX12898
1061 ACATTTCCACATTTCTGTGGAAGCCTCACAATCATT R2 0 0
2 gtHWI-EAS83_30UCEAAXX12913932 ATTAATCAACAGCA
ACATTAATCAACTGAATCAACA U0 1 0 0 chr2.fa 46078825 R
DD gtHWI-EAS83_30UCEAAXX12431647 GAATAAATAATC
AAAACATATAATACATTTTTTTAT U1 0 1 0 chr5.fa 41496935
F DD 32G gtHWI-EAS83_30UCEAAXX121412731 ATATAC
ACATATATATACATATATATATACACATAT R0 47 255 255 gtHWI-
EAS83_30UCEAAXX1213891196 GAGAAGGAAATGTGTTTTCT
AAGTTTCTTTATCTTC U1 0 1 0 chr4.fa 188020201 F DD 3
2G gtHWI-EAS83_30UCEAAXX1212641479 GTGTAGGAAAGA
AAAAAGGAGGTTGTGTAGAAAAGA U0 1 0 0 chr2.fa 19222780
4 F DD gtHWI-EAS83_30UCEAAXX1238890 TTTATTTAAAT
CTTTTAAAAANTTTTTTCCAACAAA NM 0 0 0 gtHWI-EAS83_30UC
EAAXX1213411065 GATACATATACACAAAGTAAAACTATTCAG
CCTCTA U0 1 0 0 chr17.fa 51416321 F DD gtHWI-EAS83_
30UCEAAXX121132929 GAGTTGTATTAATCTTAAATTGATAAT
TTACCATAT U1 0 1 0 chr10.fa 2376138 F DD 24G gtHWI-
EAS83_30UCEAAXX121758275 GCATTTTAACAAAATCACCAT
ATCTGGGTAACCATT U1 0 1 0 chr21.fa 27648337 R DD 18
C gtHWI-EAS83_30UCEAAXX129141000 GAAAGCACTTTATA
ATAAAACAACATTGGAGCACCT U1 0 1 0 chr8.fa 67496303 F
DD 16G
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Number of reads per Eland type

• U0 21019702 65
• U1 3280059 10
• U2 1007173 3
• R0 3661054 11
• R1 815275 2
• R2 406002 1
• NM 2050499 6
• QC 306352 1

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Peak detection

• Calculate read count at each position (bp) in
  genome
• Determine if read count is greater than expected

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Peak detection

• We need to correct for input DNA reads (control)
• - non-uniformaly distributed (form peaks too)
• - vastly different numbers of reads
  between ChIP and input

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Peak detection using ChIPseeqer
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genome
genome
T A T T A A T T A T C
C C C A T A T A T G A T A T
Expected read count total number of reads
extended fragment length / chr length
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Is the observed read count at a given genomic
position greater than expected ?
Frequency
x observed read count ? expected read count
Read count
The Poisson distribution
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Is the observed read count at a given genomic
position greater than expected ?
x 10 reads (observed) ? 0.5 reads (expected)
genome
P(Xgt10) 1.7 x 10-10
log10 P(Xgt10) -9.77
-log10 P(Xgt10) 9.77
The Poisson distribution
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Read count
Expected read count
-Log(p)
Expected read count total number of reads
extended frag len / chr len
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Read count
Expected read count
-Log(p)
Expected read count total number of reads
extended frag len / chr len
Input reads
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INPUT
ChIP
Read count
Read count
Expected read count
Expected read count
Genome positions (bp)
Genome positions (bp)
-Log(Pc)
-Log(Pi)
Threshold
Log(Pc) - Log(Pi)
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Normalized Peak score (at each bp)
P(XChIP)
R -log10
P(Xinput)
Will detect peaks with high read counts in ChIP,
low in Input Works when no input DNA !
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Non-mappable fraction of the genome
We enumerated all 30-mers, counted occurrences,
calculated non-unique fraction of genome

• chr18 9369067/76117153 0.123087459668913 (12)
• chr2 33849240/242951149 0.139325292921335
• chr3 27854877/199501827 0.139622164963933
• chr4 27090014/191273063 0.141630052737745
• chr6 24330283/170899992 0.142365618132972
• chr8 20932821/146274826 0.143106107677065
• chr5 26029902/180857866 0.143924633059643
• chr12 19382853/132349534 0.14645199279659
• chr11 20039443/134452384 0.149044906485258
• chr20 10017788/62435964 0.160449000194824
• chr7 26182588/158821424 0.164855517225434
• chr10 22968951/135374737 0.169669404417753
• chr17 14496284/78774742 0.184021980040252
• chrX 31269270/154913754 0.201849540099583
• chr1 55186693/247249719 0.223202247602959
• chr13 28668063/114142980 0.251159230291692
• chr16 23552340/88827254 0.265147676410215
• chr14 29689825/106368585 0.279122120502026
• chrM 4628/16571 0.279283084907368

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Peak detection

• Determine all genomic regions with Rgt15
• Merge peaks separated by less than 100bp
• Output all peaks with length gt 100bp
• Process 23M reads in lt7mins

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BCL6 18,814 peaks
ChIP reads
Input reads
Detected Peaks
80 are within lt20kb of a known gene
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• Where does each transcription factor bind in the
  genome, in each cell type, at a given time ? Near
  which genes ?
• What is the cis-regulatory code of each factor ?
  Does they require any co-factors ?

DNA
Activation Repression
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Regulatory Sequence Discovery using FIRE
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Discovering regulatory sequences associated with
peak regions
True TF binding peak?
Yes
Yes
Target regions
Yes
Yes
Yes
Yes

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Motif Search Algorithm
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Optimizing k-mers into more informative
degenerate motifs
True TF binding peak?
ATCCGTACA
Yes
Yes
Target regions
Yes
Yes
Yes
Yes

ATCCC/GTACA
which character increases the mutual information
by the largest amount ?
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Optimizing k-mers into more informative
degenerate motifs
True TF binding peak?
Yes
Yes
Target regions
Yes
Yes
Yes
Yes

ATCCC/GTACA
. . .
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Mutual information
change
Similarity to ChIP-chip RAP1 motif
Motif Conservation with S. bayanus
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Motifs optimized so far
k-mer MI CTCATCG 0.0618 TCATCGC
0.0485 AAAATTT 0.0438 GCTCATC 0.0434 AAAAATT
0.0383 ATGAGCT 0.0334 TTGCCAC 0.0322 TGCCACC
0.0298 ATCTCAT 0.0265 ...
MI0.081
Highly informative k-mers
MI0.045
optimize ?
Only optimize k-mer if I(k-merexpression
motif) is large enough (for all motifs optimized
so far)
Conditional mutual information I(XYZ)
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Motif co-occurrence anallysis
Discovered Motifs
Enrichment
Depletion
FIRE automatically compares discovered motifs to
known motifs in TRANSFAC and JASPAR
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ChIPseeqer an integrated framework for ChIP-seq
data analysis

• ChIPseeqer (peak detection)
• ChIPseeqer2Track (for Genome Browser)
• ChIPseeqer2FIRE ( motif analysis)
• ChIPseeqer2iPAGE ( pathway analysis)
• ChIPseeqer2cons (conservation analysis)

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Installing and setting up programs

• Install ChIPseeqer and FIRE
• http//physiology.med.cornell.edu/faculty/elemento
  /lab/chipseq.shtml
• http//tavazoielab.princeton.edu/FIRE/
• Execute following commands
• export FIREDIR/Applications/FIRE-1.1  
• export PATHPATHFIREDIR  
• export CHIPSEEQERDIR/Applications/ChIPseeqer-1.0 
   
• export PATHPATHCHIPSEEQERDIRCHIPSEEQERDIR/SC
  RIPTS
• chmod x CHIPSEEQERDIR/ChIP
• chmod x CHIPSEEQERDIR/SCRIPTS/.pl

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Peak Detection

• - Input file CTCF.bed
• cd /Desktop/elemento
• Or download from
• http//physiology.med.cornell.edu/faculty/elemento
  /lab/files/chipseq/
• - 2947043 U0 reads in BED format
• (check by typing wc l CTCF.bed)
• (view by typing more CTCF.bed and q to exit)
• - No input DNA for this experiment

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Peak Detection

• Step 1 Split big read file into one file per
  chromosome 
• split_bed_or_mit_files.pl CTCF.bed  
• Expected output
• Opening CTCF.bed
• Current directory .
• Creating ./reads.chr1

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Peak Detection

• Step 2. Detect peaks  
• ChIPseeqer --chipdir. --t15 --fraglen250
  --formatbed -outfileCTCF_peaks_t15.txt  
• Expected output
• Processing reads in chrY ... done.
• Processing reads in chrX ... done.
• Processing reads in chr9 ... done.
• Processing reads in chr8 ... done.
• Step 3. Count how many peaks were found
• wc -l CTCF_peaks_t15.txt

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Making a Genome Browser track

• Command lines
• cd JuliaChild
• wc l CTCF_peaks_t15.txt
• ChIPseeqer2track --targetsCTCF_peaks_t15.txt
  --tracknameCTCF peaks
• Expected output
• CTCF_peaks_t15.txt.wgl.gz created.
• To check that the file was created
• ls

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Making a Genome Browser track
http//genome.ucsc.edu/cgi-bin/hgGateway
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Making FIRE input files

• Command line (type instructions below as one
  single line)
• ChIPseeqer2FIRE --targetsCTCF_peaks_t15.txt
  genomewg.fa
• --suffixCTCF_peaks_t15_FIRE
• wg.fa is also available from
• http//physiology.med.cornell.edu/faculty/elemento
  /lab/files/chipseq/
• (decompress with gunzip wg.fa.gz)
• Expected output
• Extracting sequences ... Done.
• Extracting randomly selected sequences ... Done.
• CTCF_peaks_t15_FIRE.txt and CTCF_peaks_t15_FIRE.se
  q have been generated.

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FIRE analysis

• Command line (type instructions below as one
  single line)
• fire.pl --expfileCTCF_peaks_t15_FIRE.txt
• --fastafile_dnaCTCF_peaks_t15_FIRE.seq
  --nodups1 --minr2
• --specieshuman --dorna0 --dodnarna0
• Expected output
• Extracting sequences ... Done.
• Extracting randomly selected sequences ... Done.
• CTCF_peaks_t15_FIRE.txt and CTCF_peaks_t15_FIRE.se
  q have been generated.

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FIRE main output file
open CTCF_peaks_t15_FIRE.txt_FIRE/DNA/CTCF_peaks_t
15_FIRE.txt.summary.pdf
Randomly selected sequences
Peak sequences
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