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Final Results Genome Assembly Team Kelley Bullard, Henry Dewhurst, Kizee Etienne, Esha Jain, VivekSagar KR, Benjamin Metcalf, Raghav Sharma, Charles Wigington,

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Presentation on theme: "Final Results Genome Assembly Team Kelley Bullard, Henry Dewhurst, Kizee Etienne, Esha Jain, VivekSagar KR, Benjamin Metcalf, Raghav Sharma, Charles Wigington,"— Presentation transcript:

1 Final Results Genome Assembly Team Kelley Bullard, Henry Dewhurst, Kizee Etienne, Esha Jain, VivekSagar KR, Benjamin Metcalf, Raghav Sharma, Charles Wigington, Juliette Zerick

2 454 raw reads PRE-PROCESSING Illumina raw reads Pre-processing 454 reads Illumina reads Statistical analysis Read stats Published Genomes from public databases V. vulnificus YJ016 V. vulnificus CMCP6 V. vulnificus MO6-24/O Align Illumina against the reference Fastqc Prinseq NGS QC Compare mapping statistics Reference genome samstats bwa REFERENCE SELECTION Hybrid DeNovo Ray MIRA Illumina/ 454/ Hybrid DeNovo assembly 454 DeNovo Newbler CABOG SUTTA Illumina DeNovo Allpaths LG SOAP DeNovo Velvet Taipan SUTTA contigs * 3 Align illumina reads against 454 contigs Unmapped reads Mac vector CLC wb contigs Unmapped reads Evaluation GAGE Hawk-eye Illumina/(454?) reference based assembly AMOScmp contigs Unmapped reads DENOVO ASSEMBLY REFERENCE BASED ASSEMBLY Draft/ Finished genome Reference evaluation DNA Diff MUMmer Parameter optimization CONTIG MERGING All possible combinations of the best 3 Mimimus MAIA PAGIT Mauve Finished genome Scaffolds GAGE GENOME FINISHING Gap filling Nulceotide identity MUMmer GRASS Built-in Process 454 Illumina Info. Chosen Ref. Assemblers Illumina 454 LEGEND hybrid Original Pipeline

3 Read Visualization – spot the differences Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results Comparison of 454 Reads for 08-2462 (low coverage) and 2541-90 (improved coverage)

4 Read Visualization - more is better! Nav 08-2462 454 reads compared to Nav 08-2462 Illumina reads. Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

5 Read Visualization – cousins or siblings? Nav_2541-90 and Vul_06-2432 (454 and Illumina reads) coverage comparison. Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

6 Data Quality Effect of pre-processing data (using prinseq)

7 V. navarensis (454; non-preprocessed|pre-processed) Metric2423-0108-24622541-902756-81 Per Base Seq. Quality Per Seq. Quality Sc Per Base Seq. Content Per Base GC Content Per Seq. GC Content Per Base N Content Seq. Length Dist. Seq. Dup. Levels Overreprese nted Seqs. Kmer Content Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

8 V. Vulnificus (454; non-preprocessed|preprocessed) Metric 2009 V_13 68 06-243208-243508-243907-2444 Per Base Seq. Quality Per Seq. Quality Score Per Base Seq. Content Per Base GC Content Per Seq. GC Content Per Base N Content Seq. Length Dist. Seq. Dup. Levels Overrepresente d Seqs. Kmer Content Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

9 V. navarensis (Illumina; non-preprocessed|preprocessed) Metric2423-0108-24622541-902756-81 Per Base Seq. Quality Per Seq. Quality Score Per Base Seq. Content Per Base GC Content Per Seq. GC Content Per Base N Content Seq. Length Dist. Seq. Dup. Levels Overrepresented Seqs. Kmer Content Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

10 V. vulnificus (Illumina; non-preprocessed|preprocessed) Metric2009V_136806-243208-243508-243907-2444 Per Base Seq. Quality Per Seq. Quality Score Per Base Seq. Content Per Base GC Content Per Seq. GC Content Per Base N Content Seq. Length Dist. Seq. Dup. Levels Overrepresented Seqs. Kmer Content Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

11 Assembly Reference-guided and de-Novo

12 Reference guided assembly Comparison of reference guided assembly vs de-novo assembly

13 ARE – Assembly Score Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

14 Reference-guided vs de-Novo assembly ARE Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

15 Summary of Reference-guided assembly  Using V. vulnificus (CMCP6) reference strain  84% coverage  De-Novo assemblers overall provided higher assembly score than reference based assembly Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

16 De Novo Assembly Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

17 De Novo Assembly Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

18 De Novo Assembly Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

19 De Novo Assembly Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

20 De-Novo Assembler Comparison (Optimal Parameters) ARE Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

21 Final Results – V. vulnificus Graph comparing assemblers on 3 criteria: Assembly Score, Span Ratio, 1/(Break Points). Higher score for all criteria are preferable. Newbler (dn) has been removed to show variance in other tools. Span Ratio CABOG Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

22 Final Results – V. vulnificus Graph comparing assemblers on 3 criteria: Assembly Score, Span Ratio, 1/(Break Points). Higher score for all criteria are preferable. Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results 1000/(Break Points)

23 Summary of de-Novo results  OLC assemblers showed considerable differences in ARE than de-Brujin based assemblers  Cabog/Newbler vs Soap de-Novo/Velvet  Hybrid assembler, Ray, did not perform as well in terms of assembly score Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

24 Merging-Vul_06-2432 AMOScmpCABOGNewbler (dn;454) Newbler (ref;454) Newbler ref ill Ray (454)Ray(Ill)Ray (hybrid) SOAPdnVelvet AMOScmp 164.00 234.69 6.354.6963.51 55.13 64.5144.3867.22 CABOG 164.00225.12101.3062.6673.2393.8898.1175.98113.08 Newbler (dn;454) 234.69 221.89 5.48ND311.98ND419.76 104.46127.01 Newbler (ref;454) 6.35 99.30 5.48 1.4467.72 64.99 72.79 35.0772.34 Newbler (ref;Illumina) 4.6962.66 ND 1.4435.28ND Ray (454) 63.50 72.56 311.99 67.7235.2833.8149.9422.9237.68 Ray (Illumina) 55.1393.88 ND 64.99ND33.81ND Ray (hybrid) 64.51 97.17 419.76 72.79ND49.94ND SOAPdn 44.3875.98104.4635.07ND22.92ND Velvet 67.22113.08127.0172.34ND37.68ND Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

25 Merging-Nav_2541-90 AMOScmpCabogNewblerdnNewbler (ref;454) Newbler (ref;Illumina ) Ray (454)Ray (Illumina) Ray (hybrid) SOAPdnVelvet AMOScmp 133.95ND0.03 15.2614.0015.7711.2345.32 Cabog 133.95 ND 107.60 114.6082.6292.4492.5380.73123.02 Newblerdn ND 54.2159.8160.4733.1794.89 Newbler (ref;454) 0.03 107.6059.94 0.1111.611.7811.8610.1739.2 Newbler (ref;Illumina) 0.03114.60ND0.2812.6612.1512.419.639.60 Ray (454) 15.2682.6254.2111.6012.6659.1976.36 13.6563.75 Ray (Illumina) 14.0192.4459.8111.7812.1533.7924.21 11.54 39.84 Ray (hybrid) 15.7792.5360.4711.8612.4140.3336.79 14.06 ND SOAP denov o 11.2280.7333.1710.049.54 13.6111.4013.91 8.47 Velvet 45.32123.0294.8939.2039.84 64.54 39.84ND8.31 Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

26 Assembler Review AssemblerStatus454IlluminaHybridAlgorithm Allpaths LGPaired-end onlyDBG AMOScmpBB CABOGOLC MIRAZEBRA NewblerOLC RayDBG SOAPdenovoDBG SUTTAUnresolved errorsBB VelvetDBG BB = branch-and-bound; OLC = overlap consensus; DBG = de Bruijn Graph; ZEBRA Mira worked as good as our merged contigs but it is impractical – 40hr run time Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

27 454 raw reads PRE-PROCESSING Illumina raw reads Pre-processing 454 reads Illumina reads Statistical analysis Read stats Fastqc Prinseq Hybrid DeNovo Ray Mira Illumina/ 454/ Hybrid DeNovo assembly 454 DeNovo Newbler CABOG Illumina DeNovo Velvet contigs Align illumina reads against 454 contigs contigs DENOVO ASSEMBLY CONTIG MERGING Merge Ray –hyb/ Newbler Merge CABOG/Velvet MIRA-hyb Mimimus Draft genome Process 454 Illumina Info. Assemblers Illumina 454 LEGEND hybrid Final Pipeline Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

28 Splinter Pipeline 1 Pipeline 2 NUMAVGN50 Assembly Size Assembly Score Nav_2423 -0110642657.21560644.52136.53 Nav_08- 246214925736.8512303.8319.48 Nav_2541 -9016626172.51303864.3462.57 Nav_2756 -8110742939.41315914.59122.31 Vul_2009 v-13688357787.24019734.80345.03 Vul_06- 24325785122.73225254.85419.76 Vul_08- 243511142872.92303734.76144.01 Vul_08- 24399850885.72507894.99210.94 Vul_07- 24447073255.14927065.13656.10 NUMAVGN50 Assembly Size Assembly Score Nav_2423 -0112535357.01643054.42111.36 Nav_08- 2462451311.922530.140.09 Nav_2541 -9010640547.51697814.30123.02 Nav_2756 -8111141840.81321194.64124.55 Vul_2009 v-13689749705.82284084.82170.81 Vul_06- 243216728489.7783534.7632.53 Vul_08- 243519324903.72041784.8575.19 Vul_08- 243911444047.91808895.02134.64 Vul_07- 244414335905.11309425.1385.93 Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

29 Visualization Merged Newbler Ray Hybrid Pipeline / Read Processing / Assembler Results / Contig Merging / Assembler Review / Pipeline / Final Results

30 Demo

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