next gen sequencing workshop - · pdf filenext gen sequencing workshop genome quebec and...
TRANSCRIPT
Next Gen Sequencing Workshop
Genome Quebec and Mcgill Innovation Center
Maxime Caronand colleagues
[email protected]@genome.mcgill.ca
December 2012
Goals
Cover multiple types of datasets to get a broad overview of next gen sequencing
RNA-seq ChIP-seq Methyl-seq
Get familiar with compute canada clusters
Get familiar with working under linux
Cluster
Login node(s)
Execution nodes
...
1 2 3 4 5
Data
echo “command” | qsub -q qtest@mp2 -V -l walltime=1:00:0 -l nodes=1:ppn=1 -j oe -o jobs_output
qstat -u user
RNA-seq
Rna content of a cell ~ 85% rRNA ~ 10% tRNA ~ 5% mRNA, miRNA, snRNA...
Generate cDNA fragment
Map to genome considering splice sites
2 conditions, 2 replicates per condition
Fastqs (filtered)$SCRIPT setup
head -8 $MAIN/rnaseq/datasets/sample1.r1.fastq
head -8 $MAIN/rnaseq/datasets/sample1.r2.fastq
@DD63XKN1:218:D106TACXX:8:1107:6686:72194/1CGGGCGCATGCGGTCGGGGTTGTTCACTGGCTGTCCGGGGCTCCGCGCGCGTCGCCGGCCCAGCTCTGTCGCTGACGGGAGGATCTGAAGCCGGCAGA+CCCFFFFFHHHHHHIJJJJFHIIJJJJJJJJJJJJHHHFDDBBDBDDBBBB59B@BBDBBDDDB@C>C>@8ABB<ABBD9@B0>?CC@CA@BDDDD<?@DD63XKN1:218:D106TACXX:8:2305:7542:153839/1CCAGAATGTACTTCATTTTACTCTTTGACCTGCGGCCGGCTTCAGATCCTCCCGTCAGCGACAGAGCTGGGCCGGCGACGCGCGCGGAGCCCCGGACAGC+C@CFFFFFHFHHHJJJJIJJJJJGIIJGGJJIJIIJJJIIGIEIGGIJJJJIJGCHHFFFDDD@CCCDDDDDDDDDDDDDDD>BBDBBDDDDDDDBB>BA
@DD63XKN1:218:D106TACXX:8:1107:6686:72194/2GCCGGCTTCAGATCCTCCCGTCAGCGACAGAGCTGGGCCGGCGACGCGCGCGGAGCCCCGGACAGCCAGTGAACAACCCCGACCGCATGCGCCCGAGA+@C@FFFFFHGDHHJJIIIJIGIIGJIIJGGIHIFIIJJJGIIFC>?B@8B9BBDDDDDD@BDDDBBDDDCDDDCCDBBD@>BBDDDD9@CDDB@B<BB@DD63XKN1:218:D106TACXX:8:2305:7542:153839/2GAAAGACTGCCGGGCGCATGCGGTCGGGGTTGTTCACTGGCTGTCCGGGGCTCCGCGCGCGTCGCCGGCCCAGCTCTGTCGCTGACGGGGGGATCTGAAG+C@CFFFFFHHHHGJEEGHJJJJJ@FHIIJ@EHBBEFFFFEDEDADC?B@B-5<<695555&)008?5>5@D?B8:C3>>><<9>0?9@D&5>&&2>C:C@
R1
R2
Alignment$SCRIPT align
$SOFTWARE/samtools-0.1.18/samtools view $OUTDIR/alignment/sample1/accepted_hits.bam | head -4
DD63XKN1:218:D106TACXX:8:2305:20250:155750 99 chr1 41445351 255 99M = 41445453 3649CTCCAACCTCTGCGTGCGCACAGCCTAGAGCCCGCCTCCGTGAAAGACTGCCGGGCGCATGCGGTCGGGGTTGTTCACTGGCTGTCCGGGGCTCCGCGCCCCFFFFFHHHHHJJJJJJJJJHIJJJIJJJJJJIJIJJJJJJJJJJHHGHHFFDDDDDDDDDD>BDDDD>;BDBDEDDDDDDDDDDBDDD9<BBB<B@ NM:i:0 NH:i:1DD63XKN1:218:D106TACXX:8:2302:7499:179658 99 chr1 41445386 255 98M = 41445437 3598CTCCGTGAAAGACTGCCGGGCGCATGCGGTCGGGGTTGTTCACTGGCTGTCCGGGGCTCCGCGCGCGTCGCCGGCCCAGCTCTGTCGCTGACGGGAGGCCCFFFFFHHHHHJJJJJJJJJIJJIIJJFHIJJH=BEACDEEDDDDDDBDDBDDDDBBDDDD@>@>7@DBDBBB@BDBBCDCCCDDB@BBDDDD5@B NM:i:0NH:i:1DD63XKN1:218:D106TACXX:8:2204:16084:2152 97 chr1 41445386 255 53M = 41448985 3692CTCCGTGAAAGACTGCCGGGCGCATGCGGTCGGGGTTGTTCACTGGCTGTCCG =?=ADB??DFH?FHIIDAFB1CGHAAFEFFG<FGE/==3=A>AC;?=3;@@>?
NM:i:0 NH:i:1DD63XKN1:218:D106TACXX:8:2305:7542:153839 163 chr1 41445392 255 100M = 41445431 3587GAAAGACTGCCGGGCGCATGCGGTCGGGGTTGTTCACTGGCTGTCCGGGGCTCCGCGCGCGTCGCCGGCCCAGCTCTGTCGCTGACGGGGGGATCTGAAG
C@CFFFFFHHHHGJEEGHJJJJJ@FHIIJ@EHBBEFFFFEDEDADC?B@B-5<<695555&)008?5>5@D?B8:C3>>><<9>0?9@D&5>&&2>C:C@ NM:i:1NH:i:1
http://picard.sourceforge.net/explain-flags.html
Alignment (2)chr1:41444963-41478280
Alignment (3)
Cufflinks$SCRIPT fpkmknown
Quantifies transcript abundance
FPKM: fragments per kilobase per million reads
tail $OUTDIR/cufflinks_known/sample1/isoforms.fpkm_tracking
tracking_id class_code nearest_ref_id gene_id gene_short_name tss_id locus length coverage FPKM FPKM_conf_lo FPKM_conf_hi FPKM_statusENST00000331595 0 0 ENSG00000182492 BGN 0 chrX:152760396-152775012 2402 0 0 0 0 OKENST00000472615 0 0 ENSG00000182492 BGN 0 chrX:152760438-152775004 2225 0 0 0 0 OKENST00000480756 0 0 ENSG00000182492 BGN 0 chrX:152760440-152775004 2278 0 0 0 0 OKENST00000431891 0 0 ENSG00000182492 BGN 0 chrX:152760450-152772084 846 0 0 0 0 OKENST00000370204 0 0 ENSG00000182492 BGN 0 chrX:152767901-152775004 2253 0 0 0 0 OKENST00000492658 0 0 ENSG00000182492 BGN 0 chrX:152771326-152774263 420 0 0 0 0 OKENST00000451311 0 0 ENSG00000205542 TMSB4X 0 chrX:12993226-12995346 628 24435.2 1.10543e+06 1.09481e+06 1.11604e+06 OKENST00000380636 0 0 ENSG00000205542 TMSB4X 0 chrX:12993228-12995345 1702 338.059 15293.5 14619.9 15967.1 OKENST00000380635 0 0 ENSG00000205542 TMSB4X 0 chrX:12993362-12995346 767 16.3318 738.836 455.941 1021.73 OKENST00000380633 0 0 ENSG00000205542 TMSB4X 0 chrX:12993776-12995200 495 19445.9 879714 867473 891954 OK
Cuffdiff$SCRIPT cuffdiffknown
$SCRIPT parse
head $OUTDIR/cuffdiff_known/isoforms.fpkm.filtered.csv | tail -5
Differential transcript expression analysis
For differential gene expression, use deseq or edger, on raw read counts
test_id gene_id gene locus sample_1 sample_2 status value_1 value_2 log2(fold_change) test_stat p_value q_value significantENST00000380636 ENSG00000205542 TMSB4X chrX:12993226-12995346 q1 q2 OK 17154 51787.5 1.59406 -33.6146 0 0 yesENST00000434651 ENSG00000179344 HLA-DQB1 chr6:32627243-32636160 q1 q2 OK 40653.3 309.605 -7.0368 14.5901 0 0 yesENST00000451311 ENSG00000205542 TMSB4X chrX:12993226-12995346 q1 q2 OK 607642 1.14211e+06 0.910412 -61.6636 0 0 yesENST00000460185 ENSG00000179344 HLA-DQB1 chr6:32627243-32636160 q1 q2 OK 48842.6 15231.1 -1.68112 14.6929 0 0 yesENST00000464283 ENSG00000171793 CTPS1 chr1:41445006-41478235 q1 q2 OK 3728.67 13545.7 1.8611 -11.1109 0 0 yes
Cuffdiff (2)chr6:32627243-32636160
Cuffdiff (3)chrX:12993226-12995346
Cufflinks de novo$SCRIPT fpkmdenovo
Does not use an annotation reference to build transcripts
head -6 $OUTDIR/cufflinks_denovo/sample1/isoforms.fpkm_tracking
tracking_id gene_id locus length coverage FPKM FPKM_conf_lo FPKM_conf_hi FPKM_statusCUFF.1.1 CUFF.1 chr1:41445350-41478307 2927 37.6434 1692.15 1525.79 1858.51 OKCUFF.1.2 CUFF.1 chr1:41445350-41478307 2830 7.69887 346.081 255.794 436.367 OKCUFF.1.3 CUFF.1 chr1:41446049-41478307 2960 7.88763 354.566 264.404 444.728 OKCUFF.2.1 CUFF.2 chr6:32627320-32634500 1594 1541.9 70446.8 69114.3 71779.3 OKCUFF.5.1 CUFF.5 chr1:186283127-186310627 3425 102.555 4582.83 4304.65 4861.01 OKCUFF.5.2 CUFF.5 chr1:186283127-186344435 7342 212.951 9516.05 9287.23 9744.86 OK
Cuffmerge$SCRIPT merge
Merge every sample's transcript gtf by guiding to known reference
chr1 Cufflinks exon 11869 12227 . + . gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "1"; gene_name "DDX11L1"; oId "ENST00000456328"; nearest_ref "ENST00000456328"; class_code "="; tss_id "TSS1";chr1 Cufflinks exon 12613 12721 . + . gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "2"; gene_name "DDX11L1"; oId "ENST00000456328"; nearest_ref "ENST00000456328"; class_code "="; tss_id "TSS1";chr1 Cufflinks exon 13221 14409 . + . gene_id "XLOC_000001"; transcript_id "TCONS_00000001"; exon_number "3"; gene_name "DDX11L1"; oId "ENST00000456328"; nearest_ref "ENST00000456328"; class_code "="; tss_id "TSS1";
Priority Code Description1 = Complete match of intron chain2 c Contained3 j Potentially novel isoform (fragment): at least one splice junction is shared with a reference transcript4 e Single exon transfrag overlapping a reference exon and at least 10 bp of a reference intron, indicating a possible pre-mRNA fragment.5 i A transfrag falling entirely within a reference intron6 o Generic exonic overlap with a reference transcript7 p Possible polymerase run-on fragment (within 2Kbases of a reference transcript)8 r Repeat. Currently determined by looking at the soft-masked reference sequence and applied to transcripts where at least 50% 9 u Unknown, intergenic transcript10 x Exonic overlap with reference on the opposite strand11 s An intron of the transfrag overlaps a reference intron on the opposite strand (likely due to read mapping errors)12 . (.tracking file only, indicates multiple classifications)
head -3 $OUTDIR/merge_denovo/merged.gtf
De novo transcripts
De novo transcripts (2)
De novo transcript differential expression$SCRIPT cuffdiffdenovo
Runs on the previously merged gtf annotation
file (containing known and denovo transcripts)awk '$2=="j"' $OUTDIR/cuffdiff_denovo/isoforms.fpkm_tracking
tracking_id class_code nearest_ref_id gene_short_name locus length q1_FPKM q1_conf_lo q1_conf_hi q1_status q2_FPKM q2_conf_lo q2_conf_hi q2_statusTCONS_00002226 j ENST00000372621 CTPS1 chr1:41445006-41478307 3174 0 0 0 OK 0 0 0 OKTCONS_00002227 j ENST00000372621 CTPS1 chr1:41445006-41478307 3526 454.297 334.779 573.816 OK 1556.33 1240.52 1872.14 OKTCONS_00002230 j ENST00000372621 CTPS1 chr1:41445006-41478307 3230 0 0 0 OK 0 0 0 OKTCONS_00002239 j ENST00000372621 CTPS1 chr1:41445006-41478307 2162 817.073 630.149 1004 OK 3584.54 3038.53 4130.56 OKTCONS_00006887 j ENST00000271588 HMCN1 chr1:185703682-186160085 16079 4.62812 0 9.66312 OK 0 0 0 OKTCONS_00006891 j ENST00000271588 HMCN1 chr1:185703682-186160085 14027 5.981 0.207797 11.7542 OK 4.73078 0 12.3982 OKTCONS_00006907 j ENST00000287859 C1orf27 chr1:186265404-186390510 7615 281.486 234.108 328.863 OK 1301.49 1146.84 1456.13 OKTCONS_00015517 j ENST00000367478 TPR chr1:186265404-186390510 8242 0 0 0 OK 0 0 0 OKTCONS_00015518 j ENST00000367478 TPR chr1:186265404-186390510 9101 53.3195 26.6557 79.9832 OK 168.514 97.1523 239.875 OKTCONS_00015521 j ENST00000367478 TPR chr1:186265404-186390510 8679 445.843 369.391 522.296 OK 2939.97 2656.69 3223.25 OKTCONS_00015522 j ENST00000367478 TPR chr1:186265404-186390510 8955 399.175 327.324 471.027 OK 709.088 561.817 856.359 OKTCONS_00022239 j ENST00000399084 HLA-DQB1 chr6:32627144-32636160 1857 2321.15 1961.29 2681.01 OK 0 0 0 OKTCONS_00025265 j ENST00000380635 TMSB4X chrX:12993140-12995439 1222 3000.67 2587.39 3413.95 OK 9518.48 8375.59 10661.4 OK
ChIP-seq
Cross link protein to DNA
Shear DNA
Select protein bound DNA with bead-attached antibody
Sequence DNA fragments
Map to genome
Narrow or wide peaks, use an “input” (control) or not
Narrow vs Wide peaks
Peter J Park, ChIP–seq: advantages and challenges of a maturing technology, Nature, Volume 10, October 2009
Setup and fastqs
head -8 $MAIN/chipseq/datasets/treatment_narrow.r1.fastq
@HWI-ST909:110:D0PNLACXX:8:1206:5197:66384CAGGGAGTGGCACTATTAGGAGGTGTGGCCTTTTTGGAGGATGTGTGTCA+?<@DFFFDFBDFBBHBEBHIIGB<CFCHD@HEDBE<DDBGBBB?BBC8BB@HWI-ST909:110:D0PNLACXX:8:1306:14281:115130TGACACACATCCTCCAACAAGGCCACACCTCCTAATAGTGCCACTCCCTG+CCCFFFFFHGHHGJJJGIGIJJIIIJJJIJJJJJGGCGDGIJIJJJJJJJ
export SCRIPT=/home/uwy-034-aa_group/gq_workshop/Chipseq.sh
$SCRIPT setup
Alignment$SCRIPT align
$SCRIPT alignsamse
Alignment (2)
Peak calling$SCRIPT sort
$SCRIPT peakcall
head $OUTDIR/peak_calling/with_control_peaks.bed
tail -3 $OUTDIR/peak_calling/with_control_peaks.xls
Multiple peak callers
Narrow or broad peaks
chr1 120355164 120356440 MACS_peak_1 552.74chr1 120377906 120381002 MACS_peak_2 305.09
chr start end length summit tags -10*log10(pvalue) fold_enrichment FDR(%)chr1 120355165 120356440 1276 682 289 552.74 6.63 0chr1 120377907 120381002 3096 1297 459 305.09 2.58 0
Peak calling (2)
Peak calling (3)
Broad peaks
Histone modifications (Lysine methylation, acetylation, etc)
Describes different states (open/closed chromatin, actively transcribed genes, etc)
Common marks:
H3k27acH3k27me3H3k36me3H3k4me1H3k4me2H3k4me3H3k9acH3k9me3
Broad peaks (2)
Broad peaks (3)
Annotation
Annotate peak calls
Input is the .bed file of peak coordinates
Using HOMER chipseq software
PeakID Chr Start End Peak Score Annotation Distance to TSS Nearest PromoterID Nearest Ensembl Gene NameMACS_peak_1312 chr16 23107011 23110399 3156.2 promoter-TSS (NR_030705) -321 NR_030705 Snord2MACS_peak_196 chr1 195096869 195099724 3153.23 TTS (NM_008059) 1086 NM_008059 ENSMUSG00000009633 G0s2MACS_peak_1320 chr16 30063694 30068848 3147.66 exon (NM_008235, exon 3 of 4) 828 NM_008235 ENSMUSG00000022528 Hes1MACS_peak_2867 chr6 129130319 129135291 3133.91 intron (NM_053109, intron 1 of 4) 2172 NM_053109 ENSMUSG00000030157 Clec2dMACS_peak_2777 chr6 72387042 72390706 3116.9 intron (NM_145569, intron 1 of 8) 678 NM_145569 ENSMUSG00000053907 Mat2aMACS_peak_885 chr13 23830123 23832753 3100 3' UTR (NM_015786, exon 1 of 1) 762 NM_015786 ENSMUSG00000036181 Hist1h1c
Peak statistics
Motif discovery
Other
Intersection of peaks / Venn diagrams
Differential peak analysis• Count based (edgeR, DESeq)• Specific software (Diffbind, ..)
RRBS
Reduced region bisulfite sequencing~ 1/20 of CpG sites (~1 million vs ~20 million)
CpG rich regions
MspI enzyme digestion (CCGG)
Bisulfite conversion
Sequence
Protocol
Setup and fastqs
head -8 $MAIN/rrbseq/datasets/sample1.r1.fastq
@DD63XKN1:237:D1GRTACXX:2:2105:8608:109575/1TGGGGATTTGTTTTGTTAAATGTTATGGGTGTGGGGATAAGATATTGGGTTTAGAGAAAGTTTTGGGAAGAGTTTTGAGGTGGAAATAGTTTTGGTGTG+CCCFFFFFHHHHHJIIIIJJIIHJJJJJJHHIJIJJJJJFHIGGIJJIJHHIJIEGHHGHIIIHHEFBFDCECEE@B7?A;A??A>>CD>@ACB@ABBB@DD63XKN1:237:D1GRTACXX:2:2106:5775:197622/1TGGGGATTTGTTTTGTTAAATGTTATGGGTGTGGGGATAAGATATTGGGTTTAGAGAAAGTTTTGGGAAGAGTTTTGAGGTGGAAATAGTTTTGGTGT+CCCFFFFFHHHHHJJJHHIJIJHGHIJIIHIHIIJJGGIIIIIIIJIIJFGGIIBH=EHHHIIHGHFBDB@CACE@B?BD>BBDDC>CC;@AC@D<BA
head -8 $MAIN/rrbseq/datasets/sample1.r2.fastq
@DD63XKN1:237:D1GRTACXX:2:2105:8608:109575/2CAACAAAAAAACTAAAAAAACCTAAAAAATAAAAAACACCAAAAAACTATCAAAAAACCCCCACACACCAAAACTATTTCCACCTCAAAACTCTTCCCAA+BCCFFFFFHHHHGJJJIJIGJJIJJIJJIJJJJJJJJJHHHHFFFDDDDDCDDDDDDDDDDDBDDDDD?BDBABDCCDDD:CCBD<CCDDBDDDDDDCCA@DD63XKN1:237:D1GRTACXX:2:2106:5775:197622/2CAACAAAAAAACTAAAAAAACCTAAAAAATAAAAAACACCAAAAAACTATCAAAAAACCCCCACACACCAAAACTATTTCCACCTCAAAACTCTTCCCAA+CCCFFFFFHHGHHIJIJJJJJJJJJJJJJIIHIJJJIJHHHHFFFDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDEE@@DDBB<CD4A?CDCDCDDCC
export SCRIPT=/home/uwy-034-aa_group/gq_workshop/Rrbseq.sh
$SCRIPT setup
Alignment$SCRIPT align
Alignment (2)
Methylation calls$SCRIPT methylation
head $OUTDIR /methylation/sample1/sample1.map
chr pos context ratio total_C methy_C CI_lower CI_upperchr8 11540442 ACCGG 0 3 0 0 0.562chr8 11540448 GACCT 0 3 0 0 0.562chr8 11540449 ACCTG 0 3 0 0 0.562chr8 11540452 TGCTT 0 3 0 0 0.562chr8 11540464 TGCTA 0 3 0 0 0.562chr8 11540479 GACAA 0 3 0 0 0.562chr8 11540486 TACTG 0 3 0 0 0.562
Differential methylation region$SCRIPT bedgraph
$SCRIPT format
Differential methylation region (2)$SCRIPT dmr
head $OUTDIR/methylation/dmr_results.csv
id chr start end strand pvalue qvalue meth.diffchr8.11550374 chr8 11550374 11550374 0 0.0011218041 8.39004886057458e-18 23.8095238095chr8.11565909 chr8 11565909 11565909 0 0.000153455 2.06586098042011e-18 23.6363636364chr8.11565918 chr8 11565918 11565918 0 0.000153455 2.06586098042011e-18 23.6363636364chr8.11565926 chr8 11565926 11565926 0 0.0005719758 4.81257640201146e-18 20.9090909091chr8.11565936 chr8 11565936 11565936 0 0.0005719758 4.81257640201146e-18 20.9090909091chr8.11566011 chr8 11566011 11566011 0 0.0005719758 4.81257640201146e-18 20.9090909091chr8.11566210 chr8 11566210 11566210 0 1.14057593442183e-05 2.55913332556091e-19 31.5789473684chr8.11566226 chr8 11566226 11566226 0 1.1075566696939e-08 3.72757099876611e-22 47.3684210526chr8.11566229 chr8 11566229 11566229 0 1.1075566696939e-08 3.72757099876611e-22 47.3684210526
Differential analysis per site or per tile
Hyper or hypo methylated