CS 680: Assembly and Analysis of Sequencing Data. Fall 2012 August 21st, 2012

Transcription

1 CS 680: Assembly and Analysis of Sequencing Data Fall 2012 August 21st, 2012

2 of the Course

3 About the Course Instructor: Boucher Office: CSB 464 Office hours: 11 course website: Weekly readings Schedule of paper 4 credits (3 lecture, 1 lab): lab is the course project.

4 Goals of the Course Working at the interface of computer science and biology: New New data and new demands Real impact to main issues in biology. Opportunity to interact with algorithms, tools, data in current

5 Course Outline and to biology (week 1). Challenges in genome assembly (week 2 and 3): Importance and purpose of whole genome sequencing. First genera@on sequencing data and computa@onal approaches to assembly. Next genera@on sequencing methods and the de Bruijn graph approach. Specialized sequencing problems: single- cell sequencing, resequencing.

6 Course Outline SNPs and other structural (week 4): Why are structural important to detect? Calling SNPs with Samtools. of larger structural current method. Short sequence analysis of RNA (week 5) What is the difference between RNA and DNA? RNA- seq data analysis: tools and algorithms. Paper and project

7 Grading Course project: 50% 20% Class 10% Paper reviews: 20% The grading breakdown is found on the website.

8 Paper Each student or course will give one or two paper A list of the papers will be given out in the next lecture and you can sign up or suggest a related paper that you would like to present. All presented papers have to be approved. 50 minute presenta@on + 20 minute discussion.

9 Paper Reviews Address the following 1. What is the paper about? 2. What are the weaknesses of the paper? 3. What is an extension or follow- up study that could be done? Paper reviews have to be ed to me or turned in on paper at the beginning of each class. One to two pages in length.

10 Class The first 5 weeks are lectures and you will not be graded on class par@cipa@on. Each student and par@cipant is expected to read the paper prior to class and par@cipate in the paper discussion. Reflec@ng upon the paper reviews should make the class par@cipa@on easy.

11 Class Project This is the most important aspect about the course and therefore should involve either Non- trivial use of tools. Sofware development and The best projects can be submiged to a journal of conference. Project will be done in pairs or individually. I recommend pairing off with someone that has a different background than you do.

12 Class Project Schedule August 30: sample projects will be given out September 6: Submit your pairings. September 25: Submit a wrigen project proposal (3 to 4 pages). September 27: project proposal presenta@ons (audience should give feedback). December 6: final wrigen projects due. December 4&6: project presenta@ons.

13 Biology Basics

14 to Biology

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16 Two types of cells: Prokaryotes v.s. Eukaryotes

17 Prokaryotes and Eukaryotes, Prokaryotes No nucleus (bacteria or archaea) Their genomes are circular Prokaryotes do not have a nucleus, mitochondria, or any other membrane- bound organelles The genome in a prokaryote is held within a DNA/ protein complex in the cytosol called the nucleoid. Eukaryotes have nucleus (animal, plants, fungi) Linear genomes with mul@ple chromosomes in pairs

18 Chromosome for E.Coli (Prokaryote) Chromosomes for Human (Eukaryote)

19 Eukaryote (Animal) Cell

20 Prokaryotes and Eukaryotes, Prokaryotes Single cell No nucleus No organelles Eukaryotes Single or multi cell Nucleus Organelles One piece of circular DNA Chromosomes No mrna post transcriptional modification Exons/Introns splicing

21 Material for Life

22 DNA: The Code of Life The structure and the four genomic legers code for all living organisms Adenine, Guanine, Thymine, and Cytosine which pair A- T and C- G on complimentary strands.

23 DNA, DNA has a double helix structure which composed of sugar molecule phosphate group and a base (A,C,G,T) DNA always reads from 5 end to 3 end for transcrip@on replica@on 5 ATTTAGGCC 3 3 TAAATCCGG 5

24 DNA can replicate by splikng, and rebuilding each strand. Note that the rebuilding of each strand uses slightly different mechanisms due to the 5 3 asymmetry, but each daughter strand is an exact replica of the original strand. DNA Replica@on

25 Genotype/Phenotype To prevent confusion between genes (which are inherited) and developmental outcomes (which are not), make a dis@nc@on between the genotype and the phenotype of an organism Genotype: complete set of genes inherited by an individual Phenotype: all aspects of the individual s physiology, behavior, and ecological rela@onships

26 DNA the Makeup Genes are inherited and are expressed genotype makeup) phenotype (physical expression) On the lef, is the eye s phenotypes of green and black eye genes.

27 Two organisms whose genes differ at one locus are said to have different genotypes. A locus (loci for plural) is the specific loca@on of a gene of a DNA sequence on a chromosome. A variant of the DNA sequence at a given loca@on is called a allele. The ordered list of loci known for a par@cular genome is called a gene4c map.

28 Diploid and polyploid cells whose chromosomes have the same allele of a given gene at some locus are called homozygous, with respect to that gene (otherwise, it is heterzygous). The chromosomal locus of a gene might be wrigen "6p21.3 6: chromosome number p: posi@on on the chromosome s short arm ( p ) or long arm ( q ) 21.3: the posi@on on the arm: region 2, band 1, sub- band 3. The bands are visible under a microscope when the chromosome is stained.

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30 Genotype/Phenotype Blue eyes Phenotype: Brown eyes Recessive: bb Genotype: Dominant: Bb or BB

31 Pleiotropy: when one gene affects many different traits. Polygenic traits: when one trait is governed by genes, which maybe on the same chromosome or on different chromosomes. The effects of numerous genes on a single phenotype create a con@nuum of possible outcomes. Polygenic traits are also most suscep@ble to environmental influences.

32 Pleiotropy in humans: Phenylketonuria (PKU): A disorder that is caused by a deficiency of the enzyme phenylalanine hydroxylase, which is necessary to convert the essen@al amino acid phenylalanine to tyrosine. A defect in the single gene that codes for this enzyme therefore results in the mul@ple phenotypes associated with PKU, including mental retarda@on, eczema, and pigment defects that make affected individuals lighter skinned

33 Polygenic inheritance in humans: Countless traits Height is controlled by polygenes for skeleton height, but their effect may be affected by injury, and disease. Weight, skin color, and intelligence. Birth defects like clubfoot, clef palate, or neural tube defects are also the result of gene Complex diseases and traits have a tendency to have low heritability (tendency to be inherited) compared to single gene disorders (i.e. sickle- cell anemia, cys@c fibrosis, PKU, Hemophelia, many extremely rare gene@c disorders).

34 Some genes may be subject to selec4on, where individuals with advantages or traits tend to be more successful than their peers When these traits have a gene@c basis, selec@on can increase the prevalence of those traits, because the offspring will inherit those traits. This may correlate with the organism's ability to survive in its environment. Several different genotypes (and possibly phenotypes) may then coexist in a popula@on. In this case, their gene@c differences are called polymorphisms.

35 The simplest is the point or here, a single nucleo@de in the genome is changed (single nucleo4de polymorphisms (SNPs)) Other types of muta@ons include the following: Inser4on. A piece of DNA is inserted into the genome at a certain posi@on Dele4on. A piece of DNA is cut from the genome at a certain posi@on Inversion. A piece of DNA is cut, flipped around and then re- inserted, thereby conver@ng it into its complement Transloca4on. A piece of DNA is moved to a different posi@on. Duplica4on. A copy of a piece of DNA is inserted into the genome

36 and While can be detrimental to the affected individual, they can also in rare cases be beneficial; more frequently, neutral. Ofen have no or a negligible impact on survival and reproduc@on. Thereby muta@ons can increase the gene4c diversity of a popula@on, that is, the number of present polymorphisms. In combina@on with selec@on, this allow a species to adapt to changing environmental condi@ons and to survive in the long term.

37 Raw Sequence Data 4 bases: A, C, G, T + other (i.e. N = any, R = G or A (purine), Y = T or (pyrimidine)) kb (= kbp) = kilo base pairs = 1,000 bp Mb = mega base pairs = 1,000,000 bp Gb = giga base pairs = 1,000,000,000 bp. Size: E. Coli 4.6Mbp (4,600,000) Fish 130 Gbp (130,000,000,000) Paris japonica (Plant) 150 Gbp Human 3.2Gbp

38 Fasta File A sequence in FASTA format begins with a single- line descrip@on, followed by lines of sequence data (file extension is.fa). It is recommended that all lines of text be shorter than 80 characters in length.

39 Fastq File Typically contain 4 lines: Line 1 begins with a '@' character and is followed by a sequence iden@fier and an op#onal descrip@on. Line 2 is the sequence. Line 3 is the delimiter +, with an op@onal descrip@on. Line 4 is the quality score. file extension GATTTGGGGTTCAAAGCTTCAAAGCTTCAAAGC! +!!''*((((***+))%%% !!!++***!

40 Proteins: Primary Structure sequence: Sequence of amino acids = sequences from a 20 leger alphabet (i.e. ACDEFGHIKLMNPQRSTVWY)! Average protein has ~300 amino acids Typically stored as fasta files >gi gb AAD cytochrome b [Elephas maximus maximus]! LCLYTHIGRNIYYGSYLYSETWNTGIMLLLITMATAFMGYVLPWGQMSFWGATVITNLFSAIPYIGTNLV! EWIWGGFSVDKATLNRFFAFHFILPFTMVALAGVHLTFLHETGSNNPLGLTSDSDKIPFHPYYTIKDFLG! LLILILLLLLLALLSPDMLGDPDNHMPADPLNTPLHIKPEWYFLFAYAILRSVPNKLGGVLALFLSIVIL! GLMPFLHTSKHRSMMLRPLSQALFWTLTMDLLTLTWIGSQPVEYPYTIIGQMASILYFSIILAFLPIAGX! IENY!

41 Proteins: Secondary Structure chains fold into regular local structures Common types: alpha helix, beta sheet, turn, loop Defined by the of hydrogen bonds

42 Proteins: Structure 3D structure of a polypep@de sequence interac@ons between non- local and foreign atoms

43 Proteins: Quaternary Structure Arrangement of protein subunits

44 Genes and Proteins One gene encodes one protein and begins with start codon (e.g. ATG), then each three code one amino acid. Then a stop codon (e.g. TGA) signifies end of the gene. In the middle of a (eukaryo@c) gene, there are segments that are spliced out during transcrip@on. Introns: segments that are spliced out Exons: segments that are kept. Detec@ng the introns and exons is a task for gene finding.

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46 Conclusions

47 Challenges in Need to feel comfortable in interdisciplinary area Depend on others for primary data Need to address important biological and computer science problems

48 Basic Steps in Research 1. Data management problem: storage, transfer, Technology) 2. Data analysis problem: mapping, assembly algorithm scaling (Computer Science) 3. challenges: is not well suited for modeling errors over large number of 4. Biological hypothesis data (Life Science)

49 Basic Skills intelligence and machine learning and probability Algorithms Databases Programming Biology/Chemistry knowledge

50 Genomics: - Assembly - Detec@on of varia@on - GWAS RNA: - Gene expression - Transcriptome assembly - Pathway analysis Protein: - Mass spectrometry - Structure predic@on - Protein- Protein interac@on