Nextstrain Workshop


Trevor Bedford (@trvrb)
27 Mar 2019
AMD Training
CDC

Sequencing to reconstruct pathogen spread

Epidemic process

Sample some individuals

Sequence and determine phylogeny

Sequence and determine phylogeny

Pathogen genomes may reveal:

  • Evolution of new adaptive variants
  • Epidemic origins
  • Patterns of geographic spread
  • Animal-to-human spillover
  • Transmission chains

Influenza: Forecasting spread of new variants for vaccine strain selection

Bedford and Neher. 2018. bioRxiv.

Zika: Uncovering origins of the epidemic in the Americas

Faria et al. 2017. Nature.

Ebola: Revealing spatial spread and persistence in West Africa

Dudas et al. 2017. Nature.

MERS: Quantifying camel-to-human spillover

Dudas et al. 2018. eLife.

TB: Tracking individual transmission chains

Didelot et al. 2014. Mol Biol Evol.

Actionable inferences

Genomic analyses are mostly done in a retrospective manner

Dudas and Rambaut 2016

Key challenges to making genomic epidemiology actionable

  • Timely analysis and sharing of results critical
  • Dissemination must be scalable
  • Integrate many data sources
  • Results must be easily interpretable and queryable

Nextstrain

Project to conduct real-time molecular epidemiology and evolutionary analysis of emerging epidemics


with Richard Neher, James Hadfield, Emma Hodcroft, Tom Sibley,
John Huddleston, Colin Megill, Sidney Bell, Barney Potter,
Charlton Callender

Nextstrain is two things

  • a bioinformatics toolkit and visualization app, which can be used for a broad range of datasets
  • a collection of real-time pathogen analyses kept up-to-date on the website nextstrain.org

Nextstrain architecture

All code open source at github.com/nextstrain

Two central aims: (1) rapid and flexible phylodynamic analysis and
(2) interactive visualization

Fauna

Rethink database of virus and titer data

  • Harmonizes data from different sources
  • Integrates different types of data (serology, sequences, case details)
  • Provides an interface for downstream analysis

Augur

Build scripts to align sequences, build trees and annotate

  • Flexible build scripts to incorporate different viruses and analyses
  • Constructs time-resolved phylogenies
  • Annotates with geographic transitions and mutation events

Example augur pipeline for 1600 Ebola genomes

  • Align with MAFFT (34 min)
  • Build ML tree with RAxML (54 min)
  • Temporally resolve tree and geographic ancestry with TreeTime (16 min)
  • Total pipeline (1 hr 46 min)

Pipeline consists of Unix-like command line modules

  • Modules called via augur filter, augur tree, augur traits, etc...
  • Designed to be composable across pathogen builds
  • Uses Snakemake to define a pipeline, making steps obvious
  • Provides depedency graph for fast recomputation
  • Pathogen-specific repos give users an obvious foundation to build off of

Auspice

Web visualization of resulting trees

  • Interactive data exploration and filtering
  • Framework through React / D3
  • Connects phylogeny, geography and genotypes

Demo nextstrain.org focusing on visualization features

Todays' workshop

  • Brief background on phylodynamics
  • Focus of running augur and constructing snakefiles
  • Running auspice

Phylogeny describes evolutionary relationships

Phylogeny is usually a hypothesis based on characteristics of sampled taxa

Phylogeny implies a series of mutational events leading to observed tip states

Phylogenetic inference

"Data" is generally a sequence alignment

Phylogeny structures site patterns

Buffalo. 2015

Tree space is vast

There are (2n-3)!! rooted trees for n taxa

  • 3 taxa: 3 trees
  • 5 taxa: 105 trees
  • 10 taxa: 34,459,425 trees
  • 20 taxa: 8.2 × 1021 trees
  • 50 taxa: 2.8 × 1076 trees
  • 100 taxa: 3.3 × 10184 trees

Solution space is rugged

Types of phylogenetic inference methods

  • Distance-based (neighbor-joining, fast, heuristic)
  • Parsimony (fast, "model-free")
  • Maximum likelihood (infers model of mutation, accurate, examples: FastTree, RAxML)
  • Bayesian (like ML, but requires prior, produces estimates of uncertainty, examples: MrBayes, BEAST)

Inference is a tree topology, branch lengths and ancestral states

nextflu.org

Molecular clocks and dated phylogenies

Mutations tend to accumulate in a clock-like fashion

"Root-to-tip" plots show temporal signal

Allows conversion between branch length and time

Dated phylogenies provide real-world context

Grubaugh et al. 2017. Nature.

Inference of discrete traits

"Data" is a phylogeny and tip states

States include nucleotides, amino acids, geo locations, hosts, etc...

Model infers transition matrix and ancestral states

Rare transitions, short branches and many taxa increase confidence

Phylogeography

Nesting patterns are informative

Zika phylogeny infers an origin in northeast Brazil

Faria et al. 2017. Nature.

nextstrain.org/docs

Acknowledgements

Bedford Lab: Alli Black, John Huddleston, Barney Potter, James Hadfield,
Louise Moncla, Tom Sibley, Maya Lewinsohn, Katie Kistler

Nextstrain: Richard Neher, James Hadfield, Emma Hodcroft, Tom Sibley, John Huddleston, Sidney Bell, Barney Potter, Colin Megill, Charlton Callender