Metagenomic+tools+for+the+fungal+community+

Holly+Bik,+UC+Davis+19+October+2012+

hAp://phylosiE.wordpress.com+

Explicitly+PhylogeneLc+Approaches+Aligned+environmental+sequences+

Guide+Tree+

EvoluLonary+Placement+of+short+reads+

+++++++++

We+provide:+•  Support+for+Paired+End+(raw)+Illumina+data+•  Marker+gene+data+for+Bacteria,+Archaea,+Eukaryotes,+Viruses+

•  Taxonomy+assignments+based+on+probability+distribuLons+over+a+reference+phylogeny+

•  Complement+to+exisLng+tools+–+QIIME/VAMPs+–  Inputs/outputs+will+be+compaLble+for+use+with+other+soEware+tools+

Markers+

•  PMPROK+–+Dongying+Wu’s+Bac/Arch+markers+•  EukaryoLc+Orthologs+–+Parfrey+2011+paper+•  16S/18S+rRNA++•  Mitochondria+_+protein_coding+genes+•  Viral+Markers+–+Markov+clustering+on+genomes+•  Codon+Subtrees+–+finer+scale+taxonomy+

•  Extended+Markers+–+plasLds,+gene+families+

Reference+Marker+Genes+

The+Monkey+–+Build+Marker+Packages+

FastTree

hmmbuild (ssu-build)

Mapping'File'(sequence'name,'NCBI'taxon'ID)'

Reconcile'NCBI'taxonomy'IDs'with'phylogene?c'topology'

Execute'build_marker'mode'

Generate'unique'IDs'for'input'sequences'

Create'profile'HMMs'(or'CMs'for'rRNA'data)'using'input'sequences'

Alignment'File'(Marker'sequences'in'FASTA'format)'

Build'tree'and'collapse'topology'according'to'a'userMspecified'PD'cutoff'(e.g.'99%)''

Tree Reconciliation

Built Marker Packages

Index Marker Database

Clean'and'package'new'marker'genes'

New'marker'gene'packages'placed'into'shared'PhyloSiS'marker'directory'

Execute'index'mode'

Indexes'the'marker'databases'needed'for'LAST'and'Bow?e'

NOTE:'New'marker'packages'are'named'according'to'input'filenames'(e.g.'MarkerAlignment.fasta).'Core'marker'data'will'be'overwriXen'during'new'marker'builds'if'input'files'do'not'have'unique'names'compared'to'exis?ng'PhyloSiS'markers.'

Locally'indexed'marker'packages'will'not'interfere'with'automa?c'updates'to'PhyloSiS'core'markers'

Quan?ta?ve'metric'(minimum'hamming'distance)'used'to'match'edges'between'NCBI'taxon'tree'and'molecular'phylogeny'

PD'cutoff'

Built'PhyloSiS'Marker'package'

Tree' HMM'profile''(CMs'for'rRNA)'

Taxon'map' Representa?ve'sequences'

Alignment'

FastTree

hmmbuild (ssu-build)

Mapping'File'(sequence'name,'NCBI'taxon'ID)'

Reconcile'NCBI'taxonomy'IDs'with'phylogene?c'topology'

Execute'build_marker'mode'

Generate'unique'IDs'for'input'sequences'

Create'profile'HMMs'(or'CMs'for'rRNA'data)'using'input'sequences'

Alignment'File'(Marker'sequences'in'FASTA'format)'

Build'tree'and'collapse'topology'according'to'a'userMspecified'PD'cutoff'(e.g.'99%)''

Tree Reconciliation

Built Marker Packages

Index Marker Database

Clean'and'package'new'marker'genes'

New'marker'gene'packages'placed'into'shared'PhyloSiS'marker'directory'

Execute'index'mode'

Indexes'the'marker'databases'needed'for'LAST'and'Bow?e'

NOTE:'New'marker'packages'are'named'according'to'input'filenames'(e.g.'MarkerAlignment.fasta).'Core'marker'data'will'be'overwriXen'during'new'marker'builds'if'input'files'do'not'have'unique'names'compared'to'exis?ng'PhyloSiS'markers.'

Locally'indexed'marker'packages'will'not'interfere'with'automa?c'updates'to'PhyloSiS'core'markers'

Quan?ta?ve'metric'(minimum'hamming'distance)'used'to'match'edges'between'NCBI'taxon'tree'and'molecular'phylogeny'

PD'cutoff'

Built'PhyloSiS'Marker'package'

Tree' HMM'profile''(CMs'for'rRNA)'

Taxon'map' Representa?ve'sequences'

Alignment'

The+Kangaroo+–+SimulaLon+Data+

Select Taxa

PD on concatenated tree

Genome&Directory&Define&the&number&of&&genomes&to&pick&(default&=&10)&and&number&of&

reads&to&generate&per&file&(default&=&100,000)&

Grinder&algorithm&randomly&generates&reads&from&selected&genomes,&outputs&simulated&PEAIllumina&and&454&datasets&

Execute&sim&mode&

Determines&PD&contribuFons&for&taxa&present&in&concatenated&guide&tree&in&PhyloSiH&marker&directory&

Two&separate&approaches&used:&1.  Select&some&number&of&taxa&that&contribute&

to&PD&(user&input,&default&=&10&taxa)&2.  Sample&taxa&uniformly&without&replacement&

Knockout Swaths of Taxa

Generated Simulated Reads

Simulation Marker Directory

Workflow&plugs&into&updateDB&to&remove&genomes&which&have&been&used&to&simulate&metagenome&data,&as&well&as&a&swath&of&related&taxa.&

A&new&marker&directory&is&created,&where&simulated&genomes&have&been&knocked&out&from&marker&packages.&&

Compute metrics between target and

remaining taxa

Calculated&metrics&include:&the&distance&to&nearest&neighbors,&connecFng&branch&lengths,&and&the&number&of&sampled&nodes&within&various&PD&units&of&connecFng&nodes.&

DBupdate+–+Mining+new+genomes+

Amino Acid Tree

Run PhyloSift (search + align)

Execute'

phylosi/_dbupdate.pl'

A'taxa'set'is'selected'with'a'

maxPD'cutoff'of'0.02'and'a'new'

tree'is'inferred'

EBI'

Genomes'

Infer Updated Tree

PD'metric'used'to'split'guide'tree'into'

smaller'subtrees;'subsets'of'taxa'are'

selected'such'that'no'branch'connecEng'

them'has'length'>0.X'for'some'value'of'X'

Add'new'sequences'to'marker'packages'

JGI'

Genomes'

Private'

Genomes'

NCBI'

Genomes'

Nucleotide Tree

Prune Tree

Update reference sequences with

new data

New'sequences'added'at'0.25'PD'for'amino'

acid'tree;'higher'PD'threshold'enables'

more'aggressive'searches'of'reference'

database,'since'LAST'searching'is'faster'

with'fewer'sequences.'

Reconcile'NCBI'taxonomy'IDs'with'

phylogeneEc'topologies,'for'both'

amino'acid'tree'and'codon'subtrees'

Tree Reconciliation

Codon Subtrees

Package Markers

Users’'local'marker'databases'are'automaEcally'

scanned'each'Eme'PhyloSi/'is'run'and'any'new'

updates'are'automaEcally'downloaded'if'available'

Automated Download to

PhyloSift Users

Tree+ReconciliaLon+in+PhyloSiE+

Environmental,Sequences,

Named,Taxa,

Great!,,

Not,Bad,,

Ge9ng,Tricky…,,

Tree+Placement+Fat+Tree+_+Guppy+

Marine+Metagenome+

Chemoautotrophic+bacteria+–+oxidize+ammonia+into+nitrite+

Alveolate+ProLsts+

Common+seawater+Archaea+

Tree+Placement+Tog+Tree+_+Guppy+

Marine+Metagenome+

Marine+Metagenome+

Tree+Placement+Sing+Tree+_+Guppy+

Linking+with+the+Fungal+ITS+community+

•  How+does+fungal+ITS+sequence+data+relate+to+your+project?+–  PhyloSiE+has+the+capability+to+add+any+marker+gene+reference+packages+that+are+relevant+for+specific+taxonomic+communiLes++

•  What+fungal+ITS+data+does+your+project+currently+provide+– None+–+but+we+do+mine+other+marker+genes+from+fungal+genomes+

•  What+fungal+ITS+data+is+your+project+hoping+to+provide?+– We+wouldn’t+provide+data,+but+can+work+with+users+to+increase+support+for+fungal+analyses+

•  Is+your+project+involved+with+curaLng+fungal+ITS+sequences+– No,+but+we+would+curate+alignments+and+marker+packages+of+ITS+sequences+mined+from+public+databases+

•  If+so,+what+curaLon+strategies+are+being+implemented+for+your+project?+– Alignment+filtering+and+masking,+pruning+reference+trees+

•  What+tools+for+working+with+fungal+ITS+sequences+does+your+project+currently+provide?++– None+so+far+–+but+can+be+implemented+if+given+a+reference+dataset+(e.g.+alignment)+

Linking+with+the+Fungal+ITS+community+

Linking+with+the+Fungal+ITS+community+

•  What+tools+are+you+developing+/+planning+to+develop?++– Current+focus+is+on+mulLsample+comparisons+– Gene+tree+reconciliaLon+– Probability+distribuLon+over+tree+topology+to+delimit+OTUs+(PhylogeneLc+OTUs)+

•  What+framework+of+fungal+taxonomy+does+your+project+use?++– NCBI_derived+taxonomy+(because+of+tree+mapping/reconciliaLon+issues)+

SATELLITE MEETING

Eukaryotic Metagenomics

March/April 2013 UC Davis

Acknowledgements+UC+Davis+•  Jonathan+Eisen+•  Aaron+Darling+•  Guillaume+Jospin+•  Dongying+Wu+•  David+Coil+

+PhyloSiE+SoEware+Development+on+Github:+hAps://github.com/gjospin/PhyloSiE++Google+Group+for+user+support:++hAps://groups.google.com/d/forum/phylosiE++TwiAer:+@PhyloSiE+