Comparative Genomics: Background and Strategy

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1 Comparative Genomics: Background and Strategy Parimala Devi Lori Gladley Emily Norris Dhruvi Patel Jennifer Pentz Ying Sha Yuehui Zhao

2 Meningococcal meningitis Disease caused by the bacterium Neisseria meningitidis Prominent in Sub-Saharan Africa - also known as the African meningitis belt Capable of causing epidemics Sub-Saharan African meningitis belt

3 Meningitis Vaccine Project Mission seeking to eliminate meningitis in subsaharan Africa Had lots of success in serogroup A vaccinations Increase in serogroup W in vaccinated countries Alarming because serogroup W may have the potential to cause epidemics CDC Meningitis Vaccination Project

4 Questions Is there a biological difference between epidemic and endemic strains? Can CRISPRs be used to type Neisseria meningitidis? What are the dynamics of the capsule switch between serogroups C and W?

5 Epidemic vs. Endemic Presence/absence of genes Presence/absence of antibiotic resistance genes Presence/absence of virulence genes SNP variation and distribution

6 Outline for presence/absence of genes

7 Novel online resource systematically cataloguing a comprehensive pan-genome of all microbial clades Organizes currently available draft and finished bacterial genomes into qualitycontrolled clades Provides a comprehensive non-redundant reference gene catalog Number of genomes in Neisseria meningitidis: 15 including Fam18, MC58 Number of non-redundant proteins of Neisseria Meningitidis: 2776

8 Generation of gene profile

9 Database search for endemic-specific genes 1. CARD Comprehensive antibiotic resistance database 2. VFDB Virulence Factors Database

10 Single Nucleotide Polymorphism (SNP) DNA sequence variation: when two species vary genetically by a single nucleotide Non-synonymous - change AA Synonymous - silent mutations that don t change AA

11 SNPs SNPs can affect the phenotype of an organism by altering the proteins encoded within the genome Provide an excellent opportunity to compare genes genomes within and across species Goal: Try to detect SNPs in our samples and look at SNP patterns between endemic vs. epidemic strains - look for mutually exlusive SNPs

12 SNP Detection Methods SAMTools Samtools mpileup Input format: Refrence sequence- Fasta format BAM file Output format: VCF file GATK (Genome Analysis Toolkit ) Freebayes

13 Questions Is there a biological difference between epidemic and endemic strains? Can CRISPRs be used to type Neisseria meningitidis? What are the dynamics of the capsule switch between serogroups C and W?

14 CRISPR in bacteria CRISPR: clustered regularly interspaced short palindromic repeats Prashant et al., Nature Methods, 2013 CRISPR RNAs (crrnas) Trans-activating RNA (TracrRNA) Cas9 Protospacer adjacent motif (PAM)

15 Cas9 structure Hiroshi et al., Cell, 2014

16 CRISPR Unit in Bacteria and Archaea A functional CRISPR-Cas system has two distinguishable components required for activity. Devaki Bhaya et al., Annual Review Genetics, 2011 The first recognizable feature is the CRISPR locus/array located on the genome (either chromosome or plasmid), which contains the hypervariable spacers acquired from virus or plasmid DNA. The second feature is a diverse group of Cas genes located in the vicinity of a CRISPR locus, which encode proteins (generically called Cas proteins) required for the multistep defense against invasive genetic elements.

17 Cas cascades Classfication Cas1 and Cas2 are essential genes constitute three distinct Cas cascades types. Type I: Cas3 Type II: Cas9 Bacteria N. meningitidis serogroup A Type III: Cas10 Common in Archaea Kira S. Makarova et al., Nature Reviews Microbiology, 2011

18 Major Cas proteins (Cas1-Cas10) Devaki Bhaya et al., Annual Review Genetics, 2011

19 CRISPR repeats classification Based on sequence similarity, CRISPR repeats can be classified into 12 groups. Victor Kunin et al,. Genome Biology, 2007

20 Victor Kunin et al,. Genome Biology, 2007 Four groups of CRISPR repeats clearly correspond to distinct CRISPR Cas subtypes: group 2 corresponds to subtype I-E systems, group 3 corresponds to subtype I -C systems, group 4 corresponds to subtype I -F systems, group 10 corresponds to type II systems. These four groups have the most stable operon structure. Other 8 groups are prone to recombination.

21 Comparative Biology Question Phylogenetics of CRISPR-Cas loci in N. meningitidis 1. Numbers, classifications and structures of CRISPR-Cas units. 2. Comparative analysis of Cas loci Phylogenetics of Cas cascades. 3. Comparative analysis of CRISPR loci Study evolution process of CRISPR loci of N. meningitidis based on viral infection history (prone to indicate geography classification and better classify subtypes of the same strain).

22 Strategy

23 Finding family-type for CRISPR-Cas loci in 25 strains Strain 1 Strain 2 Strain 3 Strain 25 Type I Type II Type III

24 Phylogenetic analysis of CRISPR for each strain Comparison the tree with meta-data i.e geogrphic location, time collected, epidemic/endemic, serogroups etc Test the power of using CRISPR as typing method

25 Questions Is there a biological difference between epidemic and endemic strains? Can CRISPRs be used to type Neisseria meningitidis? What are the dynamics of the capsule switch between serogroups C and W?

26 Capsule switching Was the capsule switch an independent event? What is the directionality of the capsule switch? Where is the location of the break point?

27 Was capsule switch an independent event or multiple events? Strategy: Use phylogenetics Capsule Background

28 Was capsule switch an independent event or multiple events?

29 Was capsule switch an independent event or multiple events? Just background Just capsule

30 Was capsule switch an independent event or multiple events? Independent event - capsule itself and background will have same evolutionary history Just background Just capsule

31 Was capsule switch an independent event or multiple events? Multiple events - capsule itself and background will have different evolutionary histories Just background Just capsule

32 Strategy Create tree of whole genome Create tree of background Create tree of just capsular region

33 Building whole genome trees Step 1: Finding orthologs Assemble a list of orthologous genes between our genomes using a reciprocal BLASTp approach Step 2: Perform Multiple sequence alignment Clustal Omega aligns the orthologs Step 3: Build a tree Supermatrix tree approach: concatenates all alignments and generates tree

34 Reciprocal BLAST Common method for predicting putative orthologous genes BLAST between sequence data from two species where each species is used as query and subject during the process BLAST query a against dataset B -> best hit b BLAST query b against dataset A -> best hit a If a=a then b is the best reciprocal hit and genes are said to be orthologous

35 Tree building methods Maximum parsimony - simplest tree wins

36 Tree building methods Maximum parsimony - simplest tree wins Neighbor-joining tree - distance matrix method Neighbors - two taxa that are connected by single node in unrooted tree

37 Tree building methods

38 Tree building methods Maximum parsimony - simplest tree wins Neighbor-joining tree - distance matrix method Neighbors - two taxa that are connected by single node in unrooted tree Fast - practical for analyzing large data sets Robust when bootstrapping Maximum Likelihood - want to find the tree with the highest likelihood Allows you to use statistical techniques Computationally intensive

39 What is the directionality of the capsule switch? Capsule switch occurs due to horizontal gene transfer IS elements, transposons, DNA competence genes We will also be using phylogentic studies to infer the direction of horizontal gene transfer

40 What we expect to see if C to W HGT event occured C C W W W W C C C A

41 Inferring Evolution of Strains from Multiple-Locus-Sequence-Typing (i.e. MLST)

42 MLST what?? Exploits unambiguous nucleotide variation of housekeeping genes Strains that share the same sequence type (ST) are likely to share a recent common ancestor Clonal complexes are families of ST s Advantageous because comparable methods (MLEE) use electrophoretic mobility patterns, which can be difficult to compare across laboratories

43 Epidemiology: Where does MLST fit in? Detect short term or local events Identifying how strains may be related in the event of a local outbreak PFGE can detect microvariation in local events because it utilizes rapidly evolving variation Detect long term or global events Identifying how strains causing disease in one part of the world could be related to strains causing disease more globally? MLST can detect more variation than MLEE >> enhanced resolution and detection of hyperviruent lineages that emerge However, slow diversification of these lineages may eventually make them hard to distinguish from the background of isolates.

44 How does MLST work?

45 Useful Programs Genes can be located using blastn and taking the best hit or pulled directly from the annotations Blast databases of each gene prediction file may be created with ncbi makeblastdb script Genes can then be extracted from the database files with ncbi blastdbcmd script and cat into one gene file Genes can be aligned individually in MEGA and concatenated; MEGA is great for tree-drawing PubMLST Neisseria may be used to search for alleles and STs

46 Neisseria MLST Loci 7 housekeeping genes (each~ bp) abcz- ABC transporter adk- adenylate kinase aroe- shikimate dehydrogenase fumc- Fumarate hydratase class II gdh- glutamate dehydrogenase pdhc- dihydrolipoamide acetyltransferase pgm- phosphoglucomutase

47 Neisseria MLST Hyper-virulent lineages generally have identical alleles at all loci, whereas the general population has more diverse sets of alleles Strains of the same sequence type (identical alleles at all loci) may cluster together in a phylogeny, indicating decent from a common ancestor The fact that hyperviruent serogroup W strains share the same ST has hypervirulent serogroup C ST11, indicates that a capsule switch likely occurred. The tree (s) will tell the story..stay tuned!

48 Capsule Recombination Breakpoint Want to know where the capsule switch occurred

49 Capsule Recombination Breakpoint Want to know where the capsule switch occurred

50 Breakpoint Detection Methods 1. HREfinder software Homologous Recombination Events Based on SNPs and SNP positions Apply dynamic programming algorithm to model if the changes are a result of HREs Wang W-B, Jiang T, Gardner S (2013) Detection of Homologous Recombination Events in Bacterial Genomes. PLoS ONE 8(10): e doi: /journal.pone

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53 HREfinder software Input: SNP alleles and positional information, genome sequences, phylogenetic tree Based on capsule genome Output: Phylogenetic tree with number of possible HREs Locations of HREs

54 Breakpoint Detection Methods 2. Maximum χ2 Method BLAST using sliding window against known parental serogroups Use average bit scores for parental serogroups and sum for upstream and downstream windows Compute χ2 and p-values Plot against sample genome and take local maxima as breakpoints Rishishwar L, et al Genomic Basis of a Polyagglutinating Isolate of Neisseria meningitidis. J. Bacteriol. 194:

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56 Breakpoint Detection Methods 3. RDP3 Recombination Detection Program Statistical identification of recombination events Uses multiple non-parametric recombination detection methods Does not use population genetic models and no attempt to estimate population recombination rate Examples 3SEQ:identify mosaic structure or recombination in nt sequence data 4SIS: uses chi-square value to optimize breakpoint detection based on phylogenetic clustering

57 Open rectangles are areas of recombination Height proportional to negative log of p-value Each color is distinct set of recombination events Wang, Q., Z. Shao, X. Wang, Y. Gao, M. Li, L. Xu, J. Xu, L. Wang Genetic Study of Capsular Switching between Neisseria meningitidis Sequence Type 7 Serogroup A and C Strains. Infect. Immun. 78: ,

58 Questions Is there a biological difference between epidemic and endemic strains? Can CRISPRs be used to type Neisseria meningitidis? What are the dynamics of the capsule switch between serogroups C and W?

59 Thank you!