PhyloSift is a metagenomic analysis tool that provides: - Support for paired-end Illumina data and marker genes for bacteria, archaea, eukaryotes, and viruses. - Taxonomy assignments based on probability distributions over a reference phylogeny. - Tools for building marker gene packages, simulating metagenomic data, placing environmental sequences on trees, and updating reference databases with new genomes. - The ability to work with any marker genes, including fungal ITS sequences if provided a reference dataset.

1. Metagenomic+tools+for+the+
fungal+community+
Holly+Bik,+UC+Davis+
19+October+2012+

2. hAp://phylosiE.wordpress.com+

3. Explicitly+PhylogeneLc+Approaches+
Aligned+ EvoluLonary+
environmental+ Placement+of+
sequences+ short+reads+
++++
Guide+Tree+

4. 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+

5. 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+

6. Reference+Marker+Genes+

8. specified'PD'cutoff'(e.g.'99%)''
Quan?ta?ve'metric'(minimum'
Tree Reconcile'NCBI'taxonomy'IDs'
The+Monkey+–+Build+Marker+Packages+
hamming'distance)'used'to'match'
Reconciliation
with'phylogene?c'topology'
edges'between'NCBI'taxon'tree'
and'molecular'phylogeny'
Clean'and'package'new'marker'genes'
Built Marker New'marker'gene'packages'placed'into'
Packages
shared'PhyloSiS'marker'directory'
Mapping'File' PD' Alignment'File' Execute'index'mode'
(sequence'name,'NCBI'taxon'ID)' cutoff' (Marker'sequences'in'FASTA'format)'
Locally'indexed'marker'packages'
will'not'interfere'with'automa?c'
Index Marker Indexes'the'marker'databases'needed'
NOTE:'New'marker'packages'are' for'LAST'and'Bow?e'
named'according'to'input'filenames' Execute'build_marker'mode' updates'to'PhyloSiS'core'markers' Database
(e.g.'MarkerAlignment.fasta).'Core'
marker'data'will'be'overwriXen'
during'new'marker'builds'if'input' hmmbuild Create'profile'HMMs'(or'CMs'for'
files'do'not'have'unique'names' rRNA'data)'using'input'sequences'
(ssu-build)
compared'to'exis?ng'PhyloSiS'
markers.'
Built'PhyloSiS'Marker'package'
Generate'unique'IDs'for'input'sequences'
Build'tree'and'collapse' Tree' HMM'profile''
FastTree
topology'according'to'a'userM (CMs'for'rRNA)'
specified'PD'cutoff'(e.g.'99%)''
Representa?ve'
Taxon'map'
Quan?ta?ve'metric'(minimum'
sequences'
hamming'distance)'used'to'match'
Tree Reconcile'NCBI'taxonomy'IDs'
Reconciliation
with'phylogene?c'topology'
edges'between'NCBI'taxon'tree'
and'molecular'phylogeny' Alignment'
Clean'and'package'new'marker'genes'
Built Marker New'marker'gene'packages'placed'into'
Packages
shared'PhyloSiS'marker'directory'
Execute'index'mode'
Locally'indexed'marker'packages'
will'not'interfere'with'automa?c'
Index Marker Indexes'the'marker'databases'needed'
Database
for'LAST'and'Bow?e'
updates'to'PhyloSiS'core'markers'
Built'PhyloSiS'Marker'package'

9. The+Kangaroo+–+SimulaLon+Data+
Genome&Directory&
Define&the&number&of&&genomes&to&pick&(default&=&10)&and&number&of&
reads&to&generate&per&file&(default&=&100,000)&
Execute&sim&mode&
Determines&PD&contribuFons&for&taxa&
PD on present&in&concatenated&guide&tree&
concatenated tree
in&PhyloSiH&marker&directory&
Two&separate&approaches&used:&
1. Select&some&number&of&taxa&that&contribute&
Select Taxa
to&PD&(user&input,&default&=&10&taxa)&
2. Sample&taxa&uniformly&without&replacement&
Compute metrics Calculated&metrics&include:&the&distance&to&
between target and nearest&neighbors,&connecFng&branch&
remaining taxa
lengths,&and&the&number&of&sampled&nodes&
within&various&PD&units&of&connecFng&nodes.&
Knockout Workflow&plugs&into&updateDB&to&
remove&genomes&which&have&been&used&
Swaths of Taxa
to&simulate&metagenome&data,&as&well&as&
a&swath&of&related&taxa.&
Grinder&algorithm&randomly&generates&
Generated
reads&from&selected&genomes,&outputs&
Simulated Reads
simulated&PEAIllumina&and&454&datasets&
A&new&marker&directory&is&created,&
Simulation
where&simulated&genomes&have&been&
Marker Directory
knocked&out&from&marker&packages.&&

10. DBupdate+–+Mining+new+genomes+
EBI' Private' NCBI' JGI'
Genomes' Genomes' Genomes' Genomes'
Execute'
phylosi/_dbupdate.pl'
Run PhyloSift
(search + align)
Add'new'sequences'to'marker'packages'
Infer Updated
Tree
Amino Acid Nucleotide
Tree
Tree
PD'metric'used'to'split'guide'tree'into'
A'taxa'set'is'selected'with'a' Codon smaller'subtrees;'subsets'of'taxa'are'
maxPD'cutoff'of'0.02'and'a'new' Prune Tree
tree'is'inferred' Subtrees
selected'such'that'no'branch'connecEng'
them'has'length'>0.X'for'some'value'of'X'
New'sequences'added'at'0.25'PD'for'amino'
acid'tree;'higher'PD'threshold'enables' Update reference
more'aggressive'searches'of'reference' sequences with
database,'since'LAST'searching'is'faster' new data
with'fewer'sequences.'
Reconcile'NCBI'taxonomy'IDs'with'
Tree phylogeneEc'topologies,'for'both'
Reconciliation
amino'acid'tree'and'codon'subtrees'
Package
Markers
Automated
Users’'local'marker'databases'are'automaEcally'
Download to
scanned'each'Eme'PhyloSi/'is'run'and'any'new'
PhyloSift Users
updates'are'automaEcally'downloaded'if'available'

11. Tree+ReconciliaLon+in+PhyloSiE+
Environmental, Named,
Sequences, Taxa,

13. Great!,,
Not,Bad,,
Ge9ng,Tricky…,,

14. Tree+Placement+
Fat+Tree+_+Guppy+

15. Chemoautotrophic+
Marine+
bacteria+–+oxidize+
Metagenome+
ammonia+into+nitrite+
Alveolate+ProLsts+
Common+seawater+
Archaea+

16. Tree+Placement+
Tog+Tree+_+Guppy+

17. Marine+
Metagenome+

18. Marine+
Metagenome+
Tree+Placement+
Sing+Tree+_+Guppy+

19. 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+

20. Linking+with+the+Fungal+ITS+community+
• 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)+

21. 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)+

22. SATELLITE
MEETING
Eukaryotic Metagenomics
March/April 2013
UC Davis

23. 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+