Bioinformatics

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Lecture 14
Bioinformatics

• Genome sequencing projects
• Hierarchical and Shotgun approaches
• Genome assembly
• TIGR Assembler
• Ensembl

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• Genome size
• Mammalian genome 3 megabase 3x109 base pairs
• How many books are needed to print the entire
  mammalian genome?
• 1,500 letter per page x 1000 pages per book x
  2000 books
• Assuming 5 cm per book this shelf is 100 meters
  long!

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Genome sequencing the problem

• Sequencing read lengths vary depending upon
  several parameters but 600 to 800 nucleotides
  correspond to a good estimate. To sequence much
  larger fragments or even whole genome,
  essentially two strategies have been designed.
• a) The hierarchical approach. Depending on the
  vector used for cloning BAC, YAC, cosmid and
  other libraries of cloned contigs are usually
  created. The size of insert/contig may vary from
  tens to hundred thousand of base pairs.
  Collections of sub-fragments obtained by
  enzymatic restriction are mapped to get a unique
  contigs from which a minimal set of sub-fragments
  can be selected and sequenced thus limiting
  sequence redundancy.
• b) The shotgun approach. This can be applied to a
  DNA sequence of any size, including the whole
  genome. DNA is randomly fragmented by sonication
  or shearing. Following fragmentation and
  enzymatic end repair the DNA fragments are
  ligated to a plasmid vector and a bacterium host
  transformed to produce a library. Clones taken at
  random from the library are then sequenced from
  both end using two universal primers. At this
  stage a shotgun is characterised by its depth
  i.e. the cumulative length of sequence determined
  divided by the length of the fragment or genome
  to be sequenced. For example with an estimated
  size of 4 Mb a 10X shotgun would correspond to
  the assembly of about 60,000 reads with a mean
  size of 650 nt. The resulting sequences are
  assembled in a unique contig representing the
  whole fragment by sequence comparison using
  appropriate bio-informatic programs. The final
  stage or polishing stage corresponds to the
  elimination of gaps and other possible problems.

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Shotgun approach
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Genome assembly
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Assembly of a contiguous DNA sequences

• Sequencing projects have rapidly moved to using
  the two approaches sequentially.
• For example, the construction of a BAC map
  covering an entire genome or chromosome is
  followed by a shotgun strategy to sequence a
  minimal set of BACs.
• The change that was introduced by G. Venter was
  the size of the DNA fragment or genome that was
  directly shotguned. The possibility to increase
  the size of the shotgun projects was dependent
  upon the development of robots adapted to high
  throughput project and of bioinformatic programs
  that solve two major problems.
• One is a quantitative problem regarding the
  capacity to store, compare, retrieve millions of
  reads corresponding to billions of nucleotides.
  DB problem.
• The second problem is related to the presence of
  numerous repeat sequences that are often longer
  than the mean read length, complicating correct
  assembly. Assembly problem.

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Fragment assembly problem

• The Shortest Superstring Problem, while
  representing a challenge, is simplified
  abstraction, since it should also take into
  consideration three other difficulties.
• 1. Sequence data are not perfect and mistaken
  reads are possible.
• 2. Presence of numerous repeats. There is a
  million of 300 base pairs Alu copies and many
  other repeats. Fortunately some repeats may
  slightly differ due to mutation process.
• 3. As DNA is double-stranded, orientation of
  substrings is unknown and it is not known which
  strand should be used in the reconstruction.
• Most of fragment assembly algorithms include the
  following three steps
• Overlap. The problem is to find the best match
  between the suffix of one sequence an the prefix
  of another. The difficulties above force to use
  variation of the dynamic programming algorithm
  filtration methods
• Layout. This is the hardest step in DNA assembly,
  which becomes even more computationally demanding
  with increasing number of fragments. The most
  difficult is deciding whether two fragments with
  a good overlap really overlap or represent a
  repeat or something else.
• Consensus. This step is devoted to finding the
  most frequent character in the stringing layout
  that is constructed after the layout step is
  completed. More sophisticated algorithms align
  substrings in small windows along the layout or
  use a mosaic of the best (high probabilistic
  scores) segments from the layout.

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Genome assembly from smaller sequence fragments
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TIGR Assembler

• TIGR Assembler is an Open Source software.
• The TIGR Assembler is a sequence fragment
  assembly program building contigs from small
  sequence reads.
• It is versatile, offering a wide variety of
  options for tuning the assembly process and
  analyzing sequence data. The current assembly
  engine uses a greedy algorithm and heuristics to
  build contigs, find repeat regions, and target
  alignment regions.
• Sequence overlaps are detected and scored using a
  32-mer hash.
• Sequence alignment and merging is done using a
  Smith-Waterman dynamic programming algorithm.
• Gap penalties and score values corresponding to
  the bases and their quality values are predefined
  and hard coded into the program.

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Genome assembly contigs and suprcontigs
alignment

• It is very difficult to produce a finished
  continuous sequence having the level of
  redundancy typical for many high eukaryotes.
• Instead, a draft sequence of about 150,000
  contigs will be generated that could be combined
  to give a few thousand supercontigs.
• The production, in parallel, of a dense RH map
  will not only facilitate the assembly of the
  contigs into supercontigs, but will also make it
  possible to order the supercontigs a necessary
  step for understand genome rearrangements and
  synteny.

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85cM
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Mouse Genome sequencing and assembly

• The mouse genome is about 14 smaller than the
  human genome (2.5 Gb compared with 2.9 Gb)
  probably due to higher rate of deletions.
• Over 90 of mouse and human genomes can be
  partitioned into corresponding regions of
  conserved synteny.
• Sequencing strategy included four approaches 1)
  construction of BAC-based physical map by
  fingerprinting and sequencing the clones ends, 2)
  Whole-Genome Shotgun sequencing to 7 fold
  coverage and assembly to generate an initial
  draft, 3) hierarchical shotgun sequencing of BAC
  clones combined with WGS to create a hybrid
  WGS-BAC assembly, 4) production of finished
  sequence by using the BAC clones as template for
  direct finishing
• About 41 million reads were generated by the
  project participants, of which 33.6 million
  passed quality checks and 29.7 were paired
  (opposite end of the same clone). Clone inserts
  provide 47-fold physical coverage of the genome.
• Genome assembly were achieved using two newly
  developed programs Arachne and Phusion.
• The assembly contains 224,713 contigs, connected
  into 7,418 supercontigs. The 200 largest
  supercontigs span more that 98 of the assembled
  sequence, of which 3 is within sequence gaps.

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Ensembl An Open-Source Tool

• The Ensembl consists of two main parts
• 1) The analysis pipeline, which adds new data and
  analyses regularly to the core database. The DB
  contains DNA sequences, predicted features on the
  sequences and a complete body of evidence
  supporting these predictions. Ensembl known genes
  therefore are those predicted genes that have
  high similarity to genes confirmed by
  experimental evidence.
• 2) The API (application programming interface),
  which gives structured access to the data.
  Easiness of retrieving information in meaningful
  form makes API an extremely powerful tool. The
  initial implementation of the API is in Perl,
  built upon layer of Bio-Perl objects. Other
  implementations and languages like Java and
  Python are also in use.
• The Ensembl is based around two ideas a golden
  path (the pathway through the data containing
  nonredundant sequence) and virtual contig (contig
  determined by the user, an arbitrary region of a
  chromosome).
• NCBI and USCS web-sites contains systems similar
  to the Ensembl.

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