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

Preliminary Results

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

• 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

Meyer et al. 2008 Nature Protocols

Barcoding

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 coverage96% loci have sequence45 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 G—T—CC—C—C G—C—CC—T—T G—T—TC—C—T G—C—T

We need to maintain the correct info.

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