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phyloFlash v3.0 beta 1 by Harald Gruber-Vodicka, Elmar Pruesse, Brandon Seah

### High throughput phylogenetic screening using SSU rRNA gene(s) abundance(s)

Click on report section headers to expand, mouse-over underlined text to see explanations.

# Library name: test

## Graphical Summary

Mouseover on panels to expand details.

Read-mapping %identity of reads vs. reference database. Lower %identity hits may indicate presence of divergent taxa not represented in the database.
Insert sizes for read pairs. Distribution should generally be unimodal; more than one peak may indicate contamination from other libraries.
Proportion of reads mapped to SSU rRNA database. Typically < 1% for metagenomes, ca. 20% for metatranscriptomes without rRNA depletion or poly-A selection.
Proportion of reads assembled to full-length sequences. High proportion unassembled suggest either assembly failure or high diversity of organisms with low coverage.
Coverage evenness across eukaryotic 18S rRNA gene model from Barrnap, using Nhmmer from random subsample of mapped reads. This helps to detect contamination from tag sequencing libraries (sharp coverage peaks). For the eukaryotic model it is normal to see one or two regions with low coverage because of variable regions in the 18S rRNA gene that are not present in all organisms.
Coverage evenness across prokaryotic 16S rRNA gene model from Barrnap, using Nhmmer from random subsample of mapped reads. This helps to detect contamination from tag sequencing libraries (sharp coverage peaks).

### Tree of full-length assembled sequences (Click to expand)

Full-length assembled SSU rRNA sequences along with closest hits from SILVA database, in an alignment guide tree produced by MAFFT. This tree helps to visualize the relatedness of sequences in library to known relatives. Colored circles have areas proportional to the number of SSU rRNA reads that map to each respective sequence (re-mapping is done separately for SPAdes and EMIRGE full-length sequence sets). Click on the toggle switches to turn them on and off. Additional circle representing proportion of SSU rRNA reads that were not assembled is in lower right corner.

Color key: Full-length sequences assembled by SPAdes, Full-length sequences reconstructed by EMIRGE, Closest-matching reference sequence from SILVA database

## Input parameters

Input command | phyloFlash.pl -CPUs 8 -lib test -everything -read1 test_F.fq.gz -read2 test_R.fq.gz
Forward read file | test_F.fq.gz
Reverse read file | test_R.fq.gz
Minimum mapping identity | 70%
Working folder | /data/db/phyloFlash_dev/phyloFlash/test_002
Database used | /data/db/phyloFlash_dev/phyloFlash/119

## Results

### Mapping statistics

Input PE-reads | 60000
Mapped SSU read pairs | 59997
Mapping ratio | 99.995%
Fraction assembled | 88.455%
Detected median insert size | 562
Used insert size | 562
Insert size standard deviation | 154

### Output files

FASTA file of alignment of all full-length sequences | test.SSU.collection.alignment.fasta
FASTA file of all full-length sequences and their closest database hits | test.SSU.collection.fasta
Newick guide tree from MAFFT alignment of all full-length sequences and closest database hits | test.SSU.collection.fasta.tree
SVG graphic of guide tree from MAFFT alignment of all full-length sequences and closest database hits | test.SSU.collection.fasta.tree.svg
FASTA file of full-length SSU sequences | test.all.final.fasta
Mapping identity histogram from BBmap | test.idhistogram
Insert size histogram from BBmap | test.inserthistogram
phyloFlash report in plain text | test.phyloFlash
NTU abundances from initial mapping, in CSV format | test.phyloFlash.NTUabundance.csv
SVG graphic of taxonomic composition from initial read mapping | test.phyloFlash.NTUabundance.csv.svg
Taxonomic classification of full-length sequences, in CSV format | test.phyloFlash.extractedSSUclassifications.csv
phyloFlash report in CSV format | test.phyloFlash.report.csv
Taxonomic composition of unassembled SSU reads in CSV format | test.phyloFlash.unassembled.NTUabundance.csv
Reads (fwd) mapping to SSU rRNA database | test.test_F.fq.gz.SSU.1.fq
Reads (rev) mapping to SSU rRNA database | test.test_F.fq.gz.SSU.2.fq
SAM file of initial read mapping to SSU rRNA database | test.test_F.fq.gz.SSU.sam

### Taxonomic affiliation of SSU rRNA reads in library

Approximate overview of taxonomic composition of ALL reads, based on mapping hits to SILVA SSU rRNA database using BBmap.

NTUs observed once | 10
NTUs observed twice | 3
NTUs observed three or more times | 19
NTU Chao1 richness estimate | 35.6666666666667

Taxonomy summarized at level 4. Only displaying taxa with > 3 reads mapped.

Taxon | Reads
--- | ---
Eukaryota;Opisthokonta;Holozoa;Metazoa | 88901
Bacteria;Actinobacteria;Actinobacteria;Micrococcales | 1523
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales | 661
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales | 660
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales | 187
Bacteria;Firmicutes;Bacilli;Lactobacillales | 181
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales | 142
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales | 70
Bacteria;Acidobacteria;Acidobacteria;Subgroup 6 | 59
Bacteria;Spirochaetae;Spirochaetes;W27 | 55
Bacteria;Firmicutes;Bacilli;Bacillales | 26
Bacteria;Firmicutes;Clostridia;Clostridiales | 8
Eukaryota;Amoebozoa;Discosea;Longamoebia | 6
Bacteria;Actinobacteria;Actinobacteria;Corynebacteriales | 5
Eukaryota;Amoebozoa;Dictyostelia;Dictyostelium | 4
Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales | 4
Eukaryota;Opisthokonta;Nucletmycea;Fungi | 4

### SSU rRNA assembly-based taxa

Full-length SSU rRNA sequences assembled by SPAdes, matched to SILVA database with Vsearch.

OTU | Mapped | Cov | DB hit | Taxonomy | % ID | Alnlen | Evalue
--- | --- | --- | --- | --- | --- | --- | ---
test.PFspades_3 | 65695 | 304.197 | EU196000.1.1674 | Eukaryota;Opisthokonta;Holozoa;Metazoa;Animalia;Nematoda;Chromadorea;Rhabditidae;Caenorhabditis sp. JU727 | 100.0 | 1494 | -1
test.PFspades_1 | 1355 | 4.08069 | AY547556.1.1532 | Bacteria;Actinobacteria;Actinobacteria;Micrococcales;Microbacteriaceae;Microbacterium;Microbacterium sp. CME1 | 99.9 | 1505 | -1
test.PFspades_2 | 582 | 1.86115 | DQ213024.1.1496 | Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales;Xanthomonadaceae;uncultured;Xanthomonas sp. B05-08.04.0214 | 100.0 | 1496 | -1

### SSU rRNA reconstruction-based taxa

Full-length SSU rRNA seqeunces reconstructed by EMIRGE, matched to SILVA database by Vsearch.

OTU | Mapped | Ratio | DB hit | Taxonomy | % ID | Alnlen | Evalue
--- | --- | --- | --- | --- | --- | --- | ---
test.PFemirge_0 | 75803 | 0.981378 | EU196000.1.1674 | Eukaryota;Opisthokonta;Holozoa;Metazoa;Animalia;Nematoda;Chromadorea;Rhabditidae;Caenorhabditis sp. JU727 | 99.6 | 1680 | -1
test.PFemirge_59 | 1188 | 0.018622 | AY547556.1.1532 | Bacteria;Actinobacteria;Actinobacteria;Micrococcales;Microbacteriaceae;Microbacterium;Microbacterium sp. CME1 | 99.7 | 1508 | -1

### Taxonomic affiliation of unassembled SSU rRNA reads

Approximate overview of taxonomic composition for reads that did NOT assemble into full-length sequences, based on mapping hits to SILVA SSU rRNA database with BBmap.

Taxonomy summarized at level 4. Only displaying taxa with > 3 reads mapped.

Taxon | Reads
--- | ---
Eukaryota;Opisthokonta;Holozoa;Metazoa | 9005
Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales | 651
Bacteria;Actinobacteria;Actinobacteria;Micrococcales | 232
Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales | 187
Bacteria;Firmicutes;Bacilli;Lactobacillales | 181
Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales | 142
Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales | 88
Bacteria;Proteobacteria;Alphaproteobacteria;Rhodospirillales | 70
Bacteria;Spirochaetae;Spirochaetes;W27 | 55
Bacteria;Firmicutes;Bacilli;Bacillales | 26
Eukaryota;Amoebozoa;Dictyostelia;Dictyostelium | 4
Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales | 4
Eukaryota;Opisthokonta;Nucletmycea;Fungi | 4

### Interactive treemap of mapping-based taxonomic read classification

Navigation: Left-click to go down, right-click to go up in taxonomic hierarchy, hover to see counts.

Based on read-mapping hits to reference database, provides an approximate overview of taxonomic composition.

Drawn with Google Visualization API (terms of service)

### Please cite...

Gruber-Vodicka H., Pruesse E., Seah B.K.B. 2017. phyloFlash v3.0 beta 1. Online: https://github.com/HRGV/phyloFlash.

#### Cite dependencies when used

- Quast C. et al. 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucl. Acids Res. 41: D590-D596. doi:10.1093/nar/gks1219. Homepage
- Bushnell B. BBMap. Online: https://sourceforge.net/projects/bbmap/
- Bankevich A., et al. 2012. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J. Comput. Biol. 19 (5): 455-477. doi:10.1089/cmb.2012.0021. Homepage.
- Katoh K., Standley D.M.. 2013. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 30 (4): 772-780. doi:10.1093/molbev/mst010. Homepage
- Rognes T. et al. 2016. VSEARCH: a versatile open source tool for metagenomics. PeerJ 4: e2584. doi:10.7717/peerj.2584. Homepage
- Miller C.S. et al. 2011. EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data. Genome Biol. 12: R44. doi:10.1186/gb-2011-12-5-r44. Homepage
- Bedtools. Online: http://bedtools.readthedocs.io/en/latest/
- Seemann T. Barrnap. Online: https://github.com/tseemann/barrnap
- Wheeler T.J., Eddy S.R. 2013. nhmmer: DNA homology search with profile HMMs. Bioinformatics 29 (19): 2487-2489. doi:10.1093/bioinformatics/btt403 Homepage