Genome Assembly

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• Genome Assembly
• and Finishing
• Alla Lapidus, Ph.D.
• Associate Professor
• Fox Chase Cancer Center

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A typical Microbial (and not only) project
Sequencing
Draft assembly
Goals Completely restore genome Produce high
quality consensus
FINISHING
Annotation
Public release
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Sequencing Technology at a Glance
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Evolution of Microbial Drafts

• Sanger only
• 4x of 3kb plasmids 4x of 8kb plasmids 1x of
  fosmids
• 50k for 5MB genome draft

• Hybrid Sanger/pyrosequence/Illumina
• 4x 8kb Sanger 15 x coverage 454 shotgun 20x
  Illumina (quality improvement)
• 35k for 5MB genome draft

454 Solexa - 20x coverage 454 standard 4x
coverage 454 paired end (PE) 50x coverage
Illumina shotgun (quality improvement gaps) -
10k per 5MB genome
Solexa only - low cost too fragmented good
assembler is needed!
Solexa PacBio - low cost better sachffolding
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Process Overview
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Library Preparation - Sanger
DNA fragmentation
Random fragment DNA
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Library Preparation - new
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Assembly (assembler)

• Sanger reads only (phrap, PGA, Arachne)

• 454/Solexa (Newbler, PCAP, Velvet, ALLPATH etc)

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Draft assembly - what we get
Assembly set of contigs
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16
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Ordered sets of contigs (scaffolds)
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21
PE
Clone walk (Sanger lib)
New technologies no clones to walk off even if
you can scaffold contigs (bPCR new approach of
gap closing)
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Primer walking
Clone walk (captured gaps)
Clone A
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Why do we have gaps
What are gaps ? - Genome areas not covered by
random shotgun

• Sequencing coverage may not span all regions of
  the genome, thus producing gaps in the assembly
  colony picking
• Assembly results of the shotgun reads may produce
  misassembled regions due to repetitive sequences
  (new and old tech)
• A biased base content (this can result in failure
  to be cloned, poor stability in the chosen
  host-vector system, or inability of the
  polymerase to reliably copy the sequence)
• AT-rich DNA clones poorly in bacteria
  (cloning bias
• promoters like structures Sanger )gt
  uncaptured gaps
• GC rich DNA is difficult to PCR and to
  sequence and often
• requires the use of special chemistry gt
  captured gaps
• high AT and GC content caused by
  problematic PCR (new tech)

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Assembling repeats
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High GC sequencing problems

• The presence of small hairpins (inverted repeat
  sequences) in the
• DNA that re anneal ether during sequencing or
  electrophoresis
• resulting in failed sequencing reactions or
  unreadable electrophoresis
• results. (This can be aided by adding modifiers
  to the reaction,
• sequencing smaller clones and running gels at
  higher temperatures in
• the presence of stronger denaturants).

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Why more than one platform?

• 454 - high quality reliable skeletons of genomes
  (454 std 454 PE) correctly assembled contigs
  problems with repeats (unassembled or assembled
  in contigs outside of main scaffolds)
  homopolymer related frame shifts
• Illumina data is used to help improve the overall
  consensus quality, correct frameshifts and to
  close secondary structure related gaps not ready
  for de-novo assembly of complex genomes (too many
  gaps!)
• Sanger finishing reads fosmids larger
  repeats and templates for primer walk less cost
  effective but very useful in many cases

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454 (pyrosequence) and low GC genomes Thermotoga
lettingae TMO
Sanger based draft assembly - 55 total contigs
41 contigs gt2kb - 38GC - biased Sanger libraries
Draft assembly 454 - 2 total contigs 1 contigs
gt2kb - 454 no cloning
lt166bpgt - average length of gaps
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454 and High GC projects
Xylanimonas cellulosilytica DSM 15894 (3.8 MB
72.1 GC)
PGA assembly - 9x of 8kb 454
PGA assembly - 9x of 8kb
Assembly Total contigs Major contigs Scaffolds Misassenblies N50
PGA-8kb 210 166 4 165 41,048
PGA-8kb454 33 23 2 14 288,369
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NextGen high Quality Drafts at JGI (multiple
sequencing platforms)
Unassembled 454 reads
Solexa contig
454/Sanger contig
Fosmid ends and 454 PE
1.Pyrosequence and Sanger to obtain main ordered
and oriented part of the assembly Newbler
assembler
2. GapResolution (in house tool) to close some
(up to 40) gaps using unassembled 454 data
PGA or Newbler assemblers
3. Solexa reads to detect and correct errors in
consensus in house created tool (the Polisher)
and close gaps (Velvet)
Fosmids ends not used for microbes
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Solving gaps gapResopution tool
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Solving gaps gapResopution tool (II)
Step 3 If gap is not closed, tool designs
designs primers for sequencing reactions
Step 4 Iterate as necessary (in sub-assemblies)
http//www.jgi.doe.gov/degilbert_at_lbl.gov
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Solexa for gaps

• Velvet assembly
• Blast Velvet contigs against Newbler ends
• Use proper Velvet contigs to close gaps

Velvet contig
454 Contig
Gap
Illumina reads
Velvet contigs close gaps caused by hairpins and
secondary structures
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Low quality areas areas of potential
frameshifts
Assemblies contain low quality regions (red tags)
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Homopoymer related frameshifts
Frameshift 1 (AAAAA, should be AAAA)
homopolymers (ngt3)
Frameshift 2 (CCCC, should be CCC)
Modified from N. Ivanova (JGI)
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Polisher software for consensus quality
improvement
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Errors corrected by Solexa
Frame shift detected (454 contig)
CCTCTTTGATGGAAATGATATCTTCGAGCATCGCCTCGGGTT
TTCCATACAGAGAACCTTTGATGATGAACCGGTTGAAGATCTGCGGGTCA
AA CCTCTTTGATGGAAATAATATATTCGAGCATC
TTAGTGGAAATGATATCTTCGAGCATCGCCTC
CGAGCNTCGCCTCGGGCTTTCCCT
CGAGCATCGCCTCGGGTTCTCCATA
CACAGA
GCATCGCCTCGGGTTTTCAATACAGAGAACCT
CAGCGCCTCGGGTTTTCCATACAGAGAAC
CTT
ATCGCCTCGGGTTTTCCAGACAGAGAACCTTT
GGTTCGGGTTTTCCATACAGAGAAC
CTTTGAT
GTTTTCCATACAGAGAACATTTGATGATGAAC

GTTGTCCATACAGAGAACTTTTGATGATGAAC
TATANCATACAGAGAACCT
TTGATGATGAACC
ATTTCCAGACAGAGAACCNTTGATGATGAACC

CAAACAGAGAACCTTTGAGGATGAACCGGTTG

ACAGGGAACCTTAGATGATGAACCGGTTGAAG

ACAGAGAACCTTAGATGATGAACCGGTTGAAG

ACCGTTGATGATGAACCGGTTGAAGATCTGCG

GATGGTGAACGGGTTGAAGATCTGCGGGTCAA

GGTTTGAAGATCTGCGGGTCAAACCAGTCCTC

GGTGGAAGATCTGCGGGTAAAAC
CAGTCCTCT

GGT.GNAGAGCTGCGGGTCAAACCAGTCCTCTG

TGAAGATCTGCGGTTCAAACCAGTCCTCTCCC


GATCGGCGTGTCAAACCAGTCCTCTGCCTCGT

TCTGCGGGTCAAACCAGTACTCTGCCTC
GTTC
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So, what is Finishing?

• The process of taking a rough draft assembly
  composed of
• shotgun sequencing reads, identifying and
  resolving miss
• assemblies, sequence gaps and regions of low
  quality to
• produce a highly accurate finished DNA sequence.

Final quality
Final error rate should be less than 1 per 50
Kb. No gaps, no misassembled areas, no characters
other than ACGT
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Genome projectsArchaea Bacteria only
http//www.genomesonline.org/
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Metagenomic assembly and Finishing
The whole-genome shotgun sequencing approach was
used for a number of microbial community
projects, however useful quality control and
assembly of these data require reassessing
methods developed to handle relatively uniform
sequences derived from isolate microbes.

• Typically size of metagenomic sequencing project
  is very large
• Different organisms have different coverage.
  Non-uniform sequence coverage results in
  significant under- and over-representation of
  certain community members
• Low coverage for the majority of organisms in
  highly complex communities leads to poor (if any)
  assemblies
• Chimerical contigs produced by co-assembly of
  sequencing reads originating from different
  species.
• Genome rearrangements and the presence of mobile
  genetic elements (phages, transposons) in closely
  related organisms further complicate assembly.
• No assemblers developed for metagenomic data sets

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QC Annotation of poor quality sequence
To avoid this -make sure you use high quality
sequence -choose proper assembler
A Bioinformatician's Guide to Metagenomics .
Microbiol Mol Biol Rev. 2008 December 72(4)
557578.
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Assembly mistakes
A Bioinformatician's Guide to Metagenomics.
Microbiol Mol Biol Rev. 2008 December 72(4)
557578.
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Recommendations for metagenomic assembly

• Use Trimmer (Lucy etc) to treat reads PRIOR to
  assembly
• None of the existing assemblers designed for
  metagenomic data but assemblers like PGA work
  better with paired reads information and produce
  better assemblies.
• We currently test Newbler assembler for second
  generation sequencing 454 only and 454/Solexa
  co-assembly

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Metagenomic finishing approach
Candidatus Accumulibacter phosphatis (CAP)
Binning Which DNA fragment derived from which
phylotype? (BLAST GC read depth)
Lucy/PGA
Complete genome of Candidatus Accumulibacter
phosphatis
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Few more details read quality
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(No Transcript)
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Merged assemblies ( k31 and k51) with
minimus(Cloneview used for visualization)

• Green k31
• Purple k51

Illumina only data
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Stats for 31, 51 and merged 31-51 assemblies
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• Thank you!