Targeted next generation sequencing for population
genomics and phylogenomics in Ambystomatid
salamanders
Eric M. O’Neill
David W. Weisrock
Photograph by Stephen Dalton/Animals Animals - Earth Scenes
Ambystoma tigrinum complex
Coalescent Processes
• Stochastic
• Incomplete lineage
sorting
• Gene tree
incongruence
• Capture variance
• Many loci
Degnan and Rosenberg, 2006 PLOS Genetics
Goals
• Sequence >100
independent loci from
100s of samples
– both alleles
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Population genetics
Species delimitation
Gene phylogenies
Species phylogeny
Jeremiah Smith
Past Option
• Sanger Sequencing
– expensive
– cloning or computational phasing alleles
– low throughput
454 (Roche) Next Generation Sequencing
1 million reads × 400 bp each = 400 Million bp
Barcoding
Meyer et al. 2008 Nature Protocols
Methods
• Screened ~250 EST loci across 16 representative samples
• Found >100 variable loci that amplify well at the same
temperature
• Amplified 95 loci for one individual in one plate
• 94 individuals
– 8930 amplicons
• Pooled across 95 loci for each individual
• Barcoded 94 individuals and pooled
• UKY-AGTC: 454 Libraries, emPCR, 454 sequencing
Preliminary Results
• Two test runs: 1/8th picotiter plate
– 65K + 20K sequences
• One final run: 1/4th picotiter plate
– 225K sequences
• Total ~ 300K sequences
• Coverage of about 34X per sample per locus
• Sorted >95%
1664 seqs / 95 loci = 18X coverage
96% loci have sequence
45 loci had >10X coverage
Genotyping
• Clonal amplification through emPCR
• Each sequence is derived from a single DNA strand
• Identify both alleles without bacterial cloning
Errors
• Homopolymer regions
• Single nucleotide mismatches
Automated Statistical Genotyping
Hohenlohe et al., 2010 PLOS Genetics
Genotyping
• Let n be the total number of reads per site
• Let n = n1 + n2 + n3, where ni is the read count for
each possible nucleotide at the site
• For diploid, there are 10 possible genotypes
– 4 homozygous (AA, TT, GG, CC)
– 6 heterozygous (AT, AG, AC, TG, TC, GC)
• Calculate the likelihood of each possible genotype
using a multinomial sampling distribution, which gives
the probability of observing a set of read counts
(n1,n2,n3,n4)
Likelihood of a Homozygote
Likelihood of a Heterozygote
Assigning Genotypes
• The 2 equations give the likelihoods of the two most
likely hypotheses out of 10
• Use a LRT to compare the Homo vs. Het hypotheses
(df=1)
• If the test is significant, we assign the most likely
genotype at that site for that individual
• If the test is not significant, we do not assign a
genotype
• This process tests for each SNP independently, but
we want to genotype the entire sequence
8 ways to be Het at 3 SNPs:
C—T—C
C—C—C
C—T—T
C—C—T
We need to maintain the correct info.
G—T—C
G—C—C
G—T—T
G—C—T
Desired Workflow
• 454 data received as FASTA files
• Sort by barcode
– Tommy has some code for this
• Assemble by locus (alignments)
– Currently in Geneious, what other options?
• Genotype (phase the alleles)
– Need to implement automated method
– Quality scores
• Export data as sequences for phylogenetic
analysis
• Export data as alleles for population genetic
analysis