A Roadmap to the De-novo Assembly of the Banana Slug Genome
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1 A Roadmap to the De-novo Assembly of the Banana Slug Genome Stefan Prost 1 1 Department of Integrative Biology, University of California, Berkeley, United States of America April 6th-10th, 2015
2 Outline 1 A priori Information about the Genome 2 Sequencing Strategies and Platforms 3 Sequencing Libraries 4 Raw Data Processing and Quality Assessment 5 Assembly Strategies and Tools 6 Assembly Quality Assessment 7 Further Improvement of the Assembly 8 What is a finished Assembly? 9 Downstream Processing
3 Basics De-novo Assembly Changed after Green 2001 Reference-based Mapping Wikipedia
4 Basics Genome Assembly Steps Changed after Green 2001
5 Basics Genome Assembly Steps Definition: Kmer Short, unique element of DNA sequence of length n
6 (1) A priori Information about the Genome Expected Genome Size Expected Repeat Content Expected Heterozygosity Diploid or Polyploid?
7 (1) A priori Information about the Genome Genome Size A Genome Size (C-Value) Insects Arachnoids Amphibians Reptiles Birds Fishes Mammals B C-values obtained from: )
8 (1) A priori Information about the Genome A Genome Size Genome Size (C-Value) Insects Arachnoids Amphibians Reptiles Birds Genome Size Range in Fishes Fishes Mammals B at Content (%)
9 Fishes (1) A priori Information about the Genome Mammals Genome Size and Repeat Content B Repeat Content (%) Genome Size (Gb)
10 (1) A priori Information about the Genome Genome Synteny Poelstra et al. 2014
11 (1) A priori Information about the Genome Genome Size Helpful Online Databases: Genomes Genome Sizes (C-values)
12 (1) A priori Information about the Genome Expected Genome Size Expected Repeat Content Expected Heterozygosity Diploid or Polyploid?
13 (2) Sequencing Strategies and Platforms First Generation Sequencing Sanger Sequencing Second Generation Sequencing (PCR Needed) Illumina: MiSeq & HiSeq Roche: 454 Life Science: IONtorrent & IONproton ABI: SOLiD Third Generation Sequencing (Single Molecule Sequencing) Helicos Biosciences: Heliscope Pacific Biosciences: PacBio RS II Oxford Nanopore: MinION & GridION
14 (2) Sequencing Strategies and Platforms Illumina Illumina Sequencing Metzker 2010 Metzker 2010
15 (2) Sequencing Strategies and Platforms PacBio Pacific Biosciences Eid et al. 2009
16 (2) Sequencing Strategies and Platforms Nanopore Oxford Nanopore Schadt et al. 2010; Clarke et al. 2009
17 (2) Sequencing Strategies and Platforms Nanopore First Generation Sequencing Sanger Sequencing Second Generation Sequencing (PCR Needed) Illumina: MiSeq & HiSeq Roche: 454 Life Science: IONtorrent & IONproton ABI: SOLiD Third Generation Sequencing (Single Molecule Sequencing) Helicos Biosciences: Heliscope Pacific Biosciences: PacBio RS II Oxford Nanopore: MinION & GridION
18 (3) Sequencing Libraries Illumina PE Libraries Illumina Paired-end Sequencing Libraries Illumina Homepage
19 (3) Sequencing Libraries Illumina MP Libraries Illumina Mate Pair Sequencing Libraries
20 (3) Sequencing Libraries Library Recipe Allpaths-LG Recipe Custom Recipe for 1.3Gb Bird Genome (<15% Repeats) 180bp PE - 1 lane HiSeq (30x coverage) 650bp PE - 1 lane HiSeq (30x coverage) 5kb/8kb MP - 1 lane HiSeq (20x coverage) Custom Recipe for 2.5Gb Mammal Genome (<50% Repeats) 180bp PE - 4 lanes HiSeq (50x coverage) 3kb MP - 4 lanes HiSeq (40x coverage) 8kb/15kb MP - 1 lane (10x coverage)
21 (3) Sequencing Libraries BAC and Fosmid Libraries Fosmid Vector Bacterial F-plasmid <40kb Insert Size Bacterial Artificial Chromosome (BAC) <300kb Insert size
22 (4) Raw Data Processing and Quality Assessment Read Quality Assessment Base Quality Phred Score: Q Phred = -10 log 10 P(error) e.g. a Phred score of 20 translates to a 1% error rate GC Content Sequence Duplication Levels Kmer Content... Software Tools FastQC Preqc
23 (4) Raw Data Processing and Quality Assessment Fastq Format Quality Score Encoding
24 (4) Raw Data Processing and Quality Assessment Read Quality Assessment Base Quality Phred Score: Q Phred = -10 log 10 P(error) e.g. a Phred score of 20 translates to a 1% error rate GC Content Sequence Duplication Levels Kmer Content... Software Tools FastQC Preqc
25 (4) Raw Data Processing and Quality Assessment Genome Size Estimation from Read Data G = pn(l k+1) λ k G = Genome Size pn = proportion of correct reads l = read length k = kmer length λ k = mode of the k-mer count histogram Simpson 2013, arxiv
26 (4) Raw Data Processing and Quality Assessment Error Correction Error Correction (EC) using the k-mer spectrum BLESS SGA CUDA DecGPU Euler Musket Quake Reptile EC using kmer counts RACER SHREC HiTEC HSHREC PSAEC EC using Multiple Sequence Alignment Coral Echo MyHybrid
27 (4) Raw Data Processing and Quality Assessment Error Correction
28 (4) Raw Data Processing and Quality Assessment Error Correction Error Correction (EC) using the k-mer spectrum BLESS SGA CUDA DecGPU Euler Musket Quake Reptile EC using kmer counts RACER SHREC HiTEC HSHREC PSAEC EC using Multiple Sequence Alignment Coral Echo MyHybrid
29 (4) Raw Data Processing and Quality Assessment Error Correction Adapter and Low Quality Base Trimming Skewer (Jiang et al. 2014) AdapterRemoval (Lindgreen 2012) Trimmomatic (Bolger et al. 2014) Contamination Filtering Blast Allpaths-LG Removing Low Frequency k-mers Discarding Scaffolds Shorter than 1kb
30 (5) Assembly Strategies and Tools de Bruijn graph Bridges of Koenigsberg problem (Graph Theory by Euler) Compeau et al. 2011
31 (5) Assembly Strategies and Tools de Bruijn graph Compeau et al. 2011
32 (5) Assembly Strategies and Tools de Bruijn graph Berger et al. 2013
33 (5) Assembly Strategies and Tools de Bruijn graph Overlap-layout-consensus vs. de Bruijn graph Li et al. 2011, Briefings in Functional Genomcis Overlap-layout-consensus based Tools: SGA (Simpson and Durbin 2012), Celera assembler (Myers et al. 2002) de Bruijn graph based Tools: Allpaths-LG (Gnerre et al. 2011), Abyss (Simpson et al. 2009), SOAPdenovo (Luo et al. 2012)
34 (5) Assembly Strategies and Tools Assembly Tools Short Read Assembler Allpaths-LG (Gnerre et al. 2011) Abyss (Simpson et al. 2009) SOAPdenovo (Luo et al. 2012) Platanus (Kajitani et al. 2014) SSAKE (Warren et al. 2007) SGA (Simpson and Durbin 2012) Celera assembler (Myers et al. 2002) MaSuRCA (Zimin et al. 2013) Long Read Assembler Allpaths-LG (Gnerre et al. 2011) DBG2OLC (Ye et al. 2014) PBcR - Celera Assembler (Berlin et al. 2014) Short or Long Read Scaffolder SSPACE (Boetzer et al. 2011) PBjelly (English et al. 2012)
35 (5) Assembly Strategies and Tools ABySS ABySS command Output Files little eagle-unitigs.fa little eagle-contigs.fa little eagle-scaffolds.fa Get Assembly Stats: abyss-fac little eagle-scaffolds.fa
36 (6) Assembly Quality Assessment By annotation: CEGMA (Parra et al. 2007) Using RNA transcripts: Baa.pl (Ryan 2013/4, Arxiv) De novo likelihood-based measures (LAP; Ghodsi et al. 2013) Feature Response Curve (FRC; Vezzi et al. 2012) Assembly Statistics Definition: N50 contig length also called L50 A contig N50 is calculated by first ordering every contig by length from longest to shortest. Next, starting from the longest contig, the lengths of each contig are summed, until this running sum equals one-half of the total length of all contigs in the assembly. The contig N50 of the assembly is the length of the shortest contig in this list. (Yandel and Ence 2012)
37 (6) Assembly Quality Assessment Feature Response Curve Features: Mate-pair Orientations and Separations Repeat Content by k-mer Analysis Depth-of-coverage Correlated Polymorphism in the Read Alignments Read Alignment Breakpoints
38 (6) Assembly Quality Assessment Feature Response Curve By annotation: CEGMA (Parra et al. 2007) Using RNA transcripts: Baa.pl (Ryan 2013/4, Arxiv) De novo likelihood-based measures (LAP; Ghodsi et al. 2013) Feature Response Curve (FRC; Vezzi et al. 2012) Assembly Statistics Definition: N50 contig length also called L50 A contig N50 is calculated by first ordering every contig by length from longest to shortest. Next, starting from the longest contig, the lengths of each contig are summed, until this running sum equals one-half of the total length of all contigs in the assembly. The contig N50 of the assembly is the length of the shortest contig in this list. (Yandel and Ence 2012)
39 (7) Further Improvement of the Assembly Overview Gap Filling GapCloser (Luo et al. 2012) GapFiller (Nadalin et al. 2011) Resolving Mis-Assemblies REAPR (Hunt et al. 2013) NxRepair (Murphy et al. 2014) Genome Merging Metassembler (Wences and Schatz 2015, Arxiv) GAM-NGS (Vicedomini et al. 2013)
40 (7) Further Improvement of the Assembly REAPR
41 (7) Further Improvement of the Assembly Overview Gap Filling GapCloser (Luo et al. 2012) GapFiller (Nadalin et al. 2011) Resolving Mis-Assemblies REAPR (Hunt et al. 2013) NxRepair (Murphy et al. 2014) Genome Merging Metassembler (Wences and Schatz 2015, Arxiv) GAM-NGS (Vicedomini et al. 2013)
42 (7) Further Improvement of the Assembly Assembly Merging (Wences and Schatz 2015, Arxiv)
43 (7) Further Improvement of the Assembly Overview Gap Filling GapCloser (Luo et al. 2012) GapFiller (Nadalin et al. 2011) Resolving Mis-Assemblies REAPR (Hunt et al. 2013) NxRepair (Murphy et al. 2014) Genome Merging Metassembler (Wences and Schatz 2015, Arxiv) GAM-NGS (Vicedomini et al. 2013)
44 (7) Further Improvement of the Assembly Basic Workflow Allpaths-LG ABySS SOAPdenovo2 assembly using Allpaths-LG, etc. GapCloser Gap filling to resolve diffcult regions REAPR Quality assessment and breakup of misassemblies GapCloser Gap filling to resolve misassembled areas Final Assembly
45 (8) What is a finished Assembly? What is a good assembly and when is it finished? No Finished Assemblies >100 Euchromatic Gaps in Human Genome Drosophila melongaster release 6.1 (More Centric Heterochromatic Sequences Added) Contig L50 Close to or Bigger than Average Gene Size Number of Scaffolds Should be Close to Number of Chromosomes
46 (9) Downstream Processing Downstream Processing Repeat Annotation Gene Annotation Mapping to get Diploid Genome
47 Appendix - Sequencing Platforms and Libraries Roche 454 Roche 454
48 Appendix - Sequencing Platforms and Libraries ABI SOLiD ABI SOLiD
49 Appendix - Sequencing Platforms and Libraries Helicos Helicos Kircher and Kelso 2010
50 Appendix - Sequencing Platforms and Libraries Illumina SLR Libraries TruSeq Synthetic Long-Read Libraries (Moleculo)
51 Appendix - Sequencing Platforms and Libraries Illumina SLR Libraries Moleculo PacBio
52 Appendix - Sequencing Platforms and Libraries Dovetail Genomics Chicago Library Putnam et al (ArXiv)
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