{"id":140,"date":"2015-12-22T10:05:07","date_gmt":"2015-12-22T10:05:07","guid":{"rendered":"http:\/\/www.aassie.net\/Pro\/?p=140"},"modified":"2016-09-26T09:47:43","modified_gmt":"2016-09-26T09:47:43","slug":"metagenome-part-1-getting-stuff-ready","status":"publish","type":"post","link":"https:\/\/www.aassie.net\/Pro\/metagenome-part-1-getting-stuff-ready\/","title":{"rendered":"Metagenome part 1 : Getting stuff ready"},"content":{"rendered":"[vc_row type=”in_container” full_screen_row_position=”middle” scene_position=”center” text_color=”dark” text_align=”left” overlay_strength=”0.3″][vc_column column_padding=”no-extra-padding” column_padding_position=”all” background_color_opacity=”1″ background_hover_color_opacity=”1″ width=”1\/1″ tablet_text_alignment=”default” phone_text_alignment=”default”][vc_row_inner text_align=”left”][vc_column_inner column_padding=”no-extra-padding” column_padding_position=”all” background_color_opacity=”1″ width=”1\/1″][vc_column_text]\n

I am sitting on this blog for too long. I think it is time I try to publish something useful on it. So to do so I am going to present here in a series of short articles how I handle metagenome analyses. I am not pretending to have the one and only solution, I just think it will be a nice to have it nicely documented somewhere (in case I ever forget how to do it) and if it can help someone in the same time why not.<\/p>\n[\/vc_column_text][\/vc_column_inner][\/vc_row_inner][vc_row_inner text_align=”left”][vc_column_inner column_padding=”no-extra-padding” column_padding_position=”all” background_color_opacity=”1″ width=”1\/2″][vc_column_text]This first article is going to describe what the early step of preparing raw read files before the assembly phase. I will present my process using publicly available metagenome from the Human Metagenome Project<\/a>. I have selected the first metagenome available from the tongue dorsum sample category.<\/p>\n

Let\u2019s have a look a the bacterial community of the tongue of the female participant SRS147120.<\/p>\n

This is a 100 bp paired end read library sequenced with the Illumina technology.[\/vc_column_text][\/vc_column_inner][vc_column_inner column_padding=”no-extra-padding” column_padding_position=”all” background_color_opacity=”1″ width=”1\/2″][image_with_animation image_url=”148″ alignment=”right” animation=”Grow In” box_shadow=”none” max_width=”100%”][\/vc_column_inner][\/vc_row_inner][vc_column_text]After downloading the raw reads from the HMP ftp, I extracted the reads from the bz2 format. They will be recompress on the next step to a more useful .gz format.<\/p>\n

bzip2 -d SRS147120.tar.bz2
\ntar xvf SRS147120.tar<\/code><\/p>\n

We can first have a look at the library quality, to do so we can use the FastQC<\/a> software which output us with a summary looking like this:[\/vc_column_text][image_with_animation image_url=”167″ alignment=”center” animation=”Fade In” box_shadow=”none” max_width=”100%”][vc_column_text]This indicates that overall the quality of the reads is good. On the middle of the sequence we can observe a drop in quality which is apparently due to a insertion of N. A thing to do is to clean the reads of bad quality reads and other sequencing artifacts (PhiX sequences, primers, etc\u2026) using bbduk from Brian Bushnell\u2019s tool box: BBmap<\/a>.<\/p>\n

bbduk.sh in=SRS147120\/SRS147120.denovo_duplicates_marked.trimmed.1.fastq in2=SRS147120\/SRS147120.denovo_duplicates_marked.trimmed.2.fastq ref=\/tool\/bbmap\/resources\/adapters.fa,\/tool\/bbmap\/resources\/phix_adapters.fa.gz,\/tool\/bbmap\/resources\/truseq.fa.gz trimq=2 qtrim=rl out=reads_q2.fq.gz<\/code><\/span><\/p>\n

We are inputing the forward and reverse reads with in and in2, with the ref option we ask the script to check a list of known contaminant of the illumina pipeline, trimq and qtrim options are going to trim read regions with a bad quality. Finally we are going to output the screened reads in an interleaved and gz compressed format. This is what the output looks like.<\/span> <\/span><\/p>\n

BBDuk version 35.59
\nInitial:
\nMemory: max=7448m, free=7292m, used=156m
\nAdded 2970 kmers; time: 0.088 seconds.
\nMemory: max=7448m, free=6787m, used=661m
\nInput is being processed as paired
\nStarted output streams: 0.025 seconds.
\nProcessing time: 157.832 seconds.
\nInput: 92263798 reads 9226379800 bases.
\nContaminants: 47440 reads (0.05%) 4744000 bases (0.05%)
\nLow quality discards: 0 reads (0.00%) 0 bases (0.00%)
\nResult: 92216358 reads (99.95%) 9221635800 bases (99.95%)
\nTime: 157.952 seconds.
\nReads Processed: 92263k 584.12k reads\/sec
\nBases Processed: 9226m 58.41m bases\/sec<\/code><\/p>\n

We had apparently little bad apple in the assembly (0,005%\u00a0 the early quality check of the HMP people is efficient). What we can do now is to use a RNA subunit screening tool PhyloFlash<\/a>. This tool is currently developed by one of my college: Harald Gruber-Vodicka. It allows to have a idea of what organism to expect from a metagenome\/metatranscriptomic library. This is very useful to see if you have potential contamination in your library. (For example if you have butterfly sequences in a deep sea sample, it might happen). We run the following command:<\/p>\n

phyloFlash.pl -lib tongue -read1 reads_q2.fq.gz -CPUs 5<\/code><\/p>\n

Here the lib option call the name you want to prefix the outputted files, read1 your input file (can be read compressed), and CPUs call the number of processors you want to use. (Note if you ask too many processors, the process might crash during one of the steps because it distribute a fixed memory amount between the available cpus).<\/p>\n

Having a look at the outputted file tongue.phyloFlash will tell you what taxa you can expect in your library. In our case, the metagenome seems to be host of a large diversity of different bacterial taxa. The output file is placed in the following collapsed section.[\/vc_column_text][toggles style=”default”][toggle color=”Default” title=”Section”][vc_column_text]phyloFlash v2.0 – high throughput phylogenetic screening using SSU rRNA gene(s) abundance(s)
\nLibrary name: tongue
\n—
\nForward read file reads_q2.fq.gz
\nReverse read file <NONE>
\nCurrent working directory \/opt\/extern\/bremen\/molecol\/aassie\/000_blog
\n—
\nMinimum mapping identity: 70%<\/p>\n

—
\nInput PE-reads: 92216358
\nMapped SSU read pairs: 97729
\nMapping ratio: 0.105977943739656%
\nDetected median insert size: 202
\nUsed insert size: SE mode!
\nInsert size standard deviation: 412
\n—
\nRuntime: 202.52 minutes
\nCPUs used: 5
\n—
\nRead mapping based higher taxa (NTUs) detection<\/p>\n

NTUs observed once: 70
\nNTUs observed twice: 31
\nNTUs observed three or more times: 101
\nNTU Chao1 richness estimate: 180.032258064516<\/p>\n

List of NTUs in order of abundance:
\nNTU reads
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 2453
\nBacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Caenimonas 1635
\nBacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria 1199
\nBacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces 1086
\nBacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus 905
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 7 855
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas 799
\nBacteria;Proteobacteria;Gammaproteobacteria;Legionellales;Legionellaceae;Legionella 651
\nBacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonella 621
\nBacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia 466
\nBacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus 436
\nBacteria;Actinobacteria;Actinobacteria;Micrococcales;Micrococcaceae;Rothia 389
\nBacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Stomatobaculum 384
\nBacteria;Firmicutes;Erysipelotrichia;Erysipelotrichales;Erysipelotrichaceae;Solobacterium 334
\nBacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium 251
\nBacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter 241
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella 228
\nBacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcaceae UCG-014 210
\nBacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Oribacterium 204
\nBacteria;Firmicutes;Erysipelotrichia;Erysipelotrichales;Erysipelotrichaceae;Erysipelotrichaceae UCG-007 200
\nBacteria;Actinobacteria;Actinobacteria;Streptomycetales;Streptomycetaceae;Streptomyces 123
\nBacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;Escherichia-Shigella 118
\nBacteria;Firmicutes;Clostridia;Clostridiales;Family XIII;Mogibacterium 106
\nBacteria;Tenericutes;Mollicutes;Mollicutes RF9;Firmicutes oral clone FM046 95
\nBacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Lachnoanaerobaculum 92
\nBacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Peptostreptococcus 91
\nBacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;[Eubacterium] oxidoreducens group 87
\nBacteria;Saccharibacteria;uncultured bacterium 82
\nBacteria;Firmicutes;Bacilli;Bacillales;Family XI;Gemella 81
\nBacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Megasphaera 80
\nBacteria;Firmicutes;Bacilli;Lactobacillales;Lactobacillaceae;Lactobacillus 77
\nBacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Butyrivibrio 2 75
\nBacteria;Saccharibacteria;Unknown Class;Unknown Order;Unknown Family;Candidatus Saccharimonas 75
\nBacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella 74
\nBacteria;Actinobacteria;Actinobacteria;Corynebacteriales;Mycobacteriaceae;Mycobacterium 67
\nBacteria;Firmicutes;Clostridia;Clostridiales;Family XIII;[Eubacterium] nodatum group 65
\nBacteria;Actinobacteria;Coriobacteriia;Coriobacteriales;Coriobacteriaceae;Atopobium 59
\nBacteria;Saccharibacteria;TM7 phylum sp. oral clone DR034 58
\nBacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Alysiella 57
\nBacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Selenomonas 3 54
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 6 53
\nBacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;uncultured 51
\nBacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Filifactor 47
\nBacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;uncultured 43
\nBacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga 43
\nBacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Catonella 41
\nBacteria;Firmicutes;Clostridia;Clostridiales;Family XI;Parvimonas 38
\nBacteria;Cyanobacteria;Chloroplast;Aegilops tauschii 35
\nBacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Bacillus 34
\nBacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;Salmonella 33
\nBacteria;Bacteroidetes;Cytophagia;Cytophagales;Cyclobacteriaceae;Rhodonellum 31
\nBacteria;Saccharibacteria;Candidatus Saccharibacteria oral taxon TM7x 25
\nBacteria;Saccharibacteria;TM7 phylum sp. oral clone FR058 23
\nBacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Peptoclostridium 21
\nBacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;Enterobacter 19
\nBacteria;Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Staphylococcus 17
\nBacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Bergeyella 16
\nBacteria;FirmicutesBacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;[Eubacterium] coprostanoligenes group 10
\nBacteria;Synergistetes;Synergistia;Synergistales;Synergistaceae;Fretibacterium 9
\nBacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Kingella 9
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotellaceae UCG-003 9
\nBacteria;Firmicutes;Bacilli;Lactobacillales;Aerococcaceae;Abiotrophia 8
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 9 8
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 1 8
\nBacteria;Actinobacteria;Actinobacteria;Corynebacteriales;Corynebacteriaceae;Corynebacterium 8
\nBacteria;Firmicutes;Bacilli;Bacillales;Bacillaceae;Salirhabdus 7
\nBacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Eikenella 7
\nBacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Comamonadaceae;Comamonas 7
\nBacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Vagococcus 6
\nBacteria;Actinobacteria;Acidimicrobiia;Acidimicrobiales;Acidimicrobiaceae;Illumatobacter 6
\nBacteria;Firmicutes;Bacilli;Lactobacillales;P5D1-392;uncultured bacterium 6
\nBacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;Serratia 6
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Tannerella 6
\nBacteria;Proteobacteria;Gammaproteobacteria;Cardiobacteriales;Cardiobacteriaceae;Cardiobacterium 5
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidaceae;Bacteroides 5
\nBacteria;Cyanobacteria;Chloroplast;Coccomyxa sp. LA000219 5
\nBacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Dialister 5
\nBacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Basfia 5
\nBacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Vogesella 5
\nBacteria;Proteobacteria;Betaproteobacteria;Burkholderiales;Burkholderiaceae;Lautropia 5
\nBacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Snodgrassella 4
\nBacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Bacteroidales S24-7 group;uncultured bacterium 4
\nBacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Sebaldella 4
\nBacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Actinobacillus 4
\nBacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae;Vibrio 4
\nBacteria;Saccharibacteria;candidate division TM7 bacterium JGI 0001002-L20 4
\nBacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;CFT112H7;uncultured bacterium 3
\nBacteria;Firmicutes;Bacilli;Lactobacillales;Enterococcaceae;Enterococcus 3
\nBacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Aggregatibacter 3
\nBacteria;Proteobacteria;Alphaproteobacteria;Sphingomonadales;Erythrobacteraceae;Altererythrobacter 3
\nBacteria;Proteobacteria;Gammaproteobacteria;Vibrionales;Vibrionaceae;Salinivibrio 3
\nBacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Vitreoscilla 3
\nBacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Gallibacterium 3
\nBacteria;Actinobacteria;Actinobacteria;Micrococcales;Intrasporangiaceae;Tetrasphaera 3
\nBacteria;Firmicutes;Bacilli;Lactobacillales;P5D1-392;unidentified 3
\n;Bacilli;Bacillales;Planococcaceae;Planomicrobium 16
\nBacteria;Firmicutes;Bacilli;Bacillales;Paenibacillaceae;Cohnella 13
\nBacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Lachnospiraceae UCG-008 12
\nBacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales;Pseudomonadaceae;Pseudomonas 11
\nBacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Selenomonas 11
\nBacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;ASCC02;Granulicatella sp. oral clone ASC02 10
\nBacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;[Eubacterium] coprostanoligenes group 10
\n—
\nSSU assembly based taxa:
\nOTU coverage dbHit taxonomy %id alnlen evalue
\ntongue.PFspades_1 163.282 ACQV01000002.9075.10620 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;Neisseria flavescens SK114 99.7 1523 -1 99.5 1536 1521 2 3 1518
\ntongue.PFspades_16 58.4367 HQ777184.1.1470 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured organism 100.0 1176 -1 99.9 1176 1176 0 0 1176
\ntongue.PFspades_15 49.3781 JQ449239.1.1395 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;uncultured bacterium 99.8 959 -1 99.7 964 958 1 1 957
\ntongue.PFspades_2 33.0807 AJTC01000001.52.1592 Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;Haemophilus parainfluenzae HK2019 99.6 1520 -1 99.5 1536 1520 0 6 1514
\ntongue.PFspades_5 23.7396 GQ338720.1.1529 Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonella;uncultured bacterium 98.3 1529 -1 98.2 1564 1527 2 24 1503
\ntongue.PFspades_3 20.5142 HK240338.10.1530 Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;unidentified 98.9 1521 -1 98.8 1538 1521 0 16 1505
\ntongue.PFspades_10 17.6272 ACJY01000002.122.1615 Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;Fusobacterium periodonticum ATCC 33693 99.8 1486 -1 99.7 1503 1486 0 3 1483
\ntongue.PFspades_18 10.3672 JQ461559.1.1411 Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;uncultured bacterium 100.0 957 -1 99.9 961 957 0 0 957
\ntongue.PFspades_7 9.84772 HK241843.9.1513 Bacteria;Firmicutes;Erysipelotrichia;Erysipelotrichales;Erysipelotrichaceae;Solobacterium;unidentified 99.6 1505 -1 99.5 1522 1505 0 6 1499
\ntongue.PFspades_4 9.64834 ACSB01000014.3447.4963 Bacteria;Actinobacteria;Actinobacteria;Micrococcales;Micrococcaceae;Rothia;Rothia mucilaginosa M508 99.8 1509 -1 99.7 1526 1509 0 3 1506
\ntongue.PFspades_14 8.7575 KC169767.1.1488 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;uncultured Bacteroidetes bacterium 99.8 959 -1 99.7 963 959 0 2 957
\ntongue.PFspades_8 8.19441 AF385520.1.1496 Bacteria;Saccharibacteria;TM7 phylum sp. oral clone DR034 99.5 1490 -1 99.3 1525 1490 0 8
\n1482
\ntongue.PFspades_11 6.41068 AENQ01000044.3766.5256 Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;Campylobacter concisus UNSWCD 99.9 1491 -1 99.8 1508 1491 0 1 1490
\ntongue.PFspades_9 4.48011 AWVR01000095.17.1514 Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;Leptotrichia sp. oral taxon 215 str. W9775 99.4 1490 -1 99.3 1507 1490 0 9 1481
\ntongue.PFspades_6 4.00532 AF287763.1.1537 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Peptostreptococcus;Peptostreptococcus sp. oral clone CK035 99.7 1523 -1 99.6 1541 1523 0 4 1519
\ntongue.PFspades_13 3.01362 AF385510.1.1522 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Stomatobaculum;Eubacterium sp. oral clone DO016 95.5 1514 -1 95.8 1530 1506 8 60 1446
\ntongue.PFspades_12 1.73785 HE681253.1.1518 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcaceae UCG-014;uncultured bacterium 99.5 1509 -1 99.3 1526 1509 0 8 1501
\ntongue.PFspades_17 1.65886 JQ465246.1.1375 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII;Mogibacterium;uncultured bacterium 99.9 972 -1 99.8 978 972 0 1 971
\n—
\nSSU reconstruction based taxa:
\nOTU ratio dbHit taxonomy %id alnlen evalue
\ntongue.PFemirge_7 0.196360 JQ449243.1.1398 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;uncultured bacterium 97.4 1401 -1 97.4 1401 1398 3 34 1364
\ntongue.PFemirge_31 0.058397 JQ460126.1.1415 Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonella;uncultured bacterium 99.9 1314 -1 99.9 1314 1314 0 1 1313
\ntongue.PFemirge_169 0.057802 FM995684.1.1473 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;uncultured bacterium 99.7 1473 -1 99.5 1510 1473 0 5 1468
\ntongue.PFemirge_10 0.046183 JQ449266.1.1397 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 7;uncultured bacterium 99.8 1390 -1 99.6 1417 1390 0 3 1387
\ntongue.PFemirge_350 0.042717 EF511181.1.1499 Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;uncultured bacterium 99.2 1481 -1 99.2 1481 1481 0 12 1469
\ntongue.PFemirge_572 0.030953 JQ449672.1.1409 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured bacterium 99.3 1409 -1 99.3 1409 1409 0 10 1399
\ntongue.PFemirge_88 0.022259 AJTC01000001.52.1592 Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;Haemophilus parainfluenzae HK2019 98.8 1515 -1 99.4 1515 1504 11 7 1497
\ntongue.PFemirge_93 0.021629 JQ451880.1.1378 Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;uncultured bacterium 96.7 1379 -1 96.8 1377 1375 4 41 1334
\ntongue.PFemirge_111 0.021404 AF366269.1.1534 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 7;uncultured Prevotella sp. 99.5 1517 -1 99.5 1517 1517 0 7 1510
\ntongue.PFemirge_192 0.021016 JQ460482.1.1399 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;uncultured bacterium 99.8 1399 -1 99.8 1399 1399 0 3 1396
\ntongue.PFemirge_9782 0.020839 AEVD01000030.72.1605 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;Streptococcus infantis ATCC 700779 80.4 1554 -1 83.8 1538 1487 67 238 1249
\ntongue.PFemirge_207 0.020479 KF101920.1.1362 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;uncultured bacterium 99.7 1362 -1 99.7 1362 1362 0 4 1358
\ntongue.PFemirge_25 0.019135 AF385504.1.1539 Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;Actinomyces sp. oral clone CT047 99.0 1526 -1 99.0 1526 1525 1 14 1511
\ntongue.PFemirge_15 0.016818 HK241642.1.1511 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;unidentified 99.7 1511 -1 99.7 1511 1511 0 4 1507
\ntongue.PFemirge_373 0.015839 AB547695.1.1489 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;Prevotella nanceiensis 99.4 1398 -1 99.4 1398 1398 0 8 1390
\ntongue.PFemirge_197 0.015737 JQ449272.1.1394 Bacteria;Proteobacteria;Gammaproteobacteria;Pasteurellales;Pasteurellaceae;Haemophilus;uncultured bacterium 99.8 1394 -1 99.8 1394 1394 0 3 1391
\ntongue.PFemirge_9708 0.015438 ACQV01000002.9075.10620 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;Neisseria flavescens SK114 95.8 1511 -1 95.9 1509 1509 2 61 1448
\ntongue.PFemirge_610 0.014278 HK241843.9.1513 Bacteria;Firmicutes;Erysipelotrichia;Erysipelotrichales;Erysipelotrichaceae;Solobacterium;unidentified 99.6 1505 -1 99.6 1505 1505 0 6 1499
\ntongue.PFemirge_719 0.012764 HQ778326.1.1469 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured organism 96.2 1465 -1 96.5 1461 1457 8 47 1410
\ntongue.PFemirge_180 0.012558 KF098306.1.1368 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured bacterium 99.0 1368 -1 99.0 1368 1368 0 13 1355
\ntongue.PFemirge_1166 0.012404 AF385520.1.1496 Bacteria;Saccharibacteria;TM7 phylum sp. oral clone DR034 99.5 1490 -1 99.5 1490 1490 0
\n7 1483
\ntongue.PFemirge_9825 0.011649 AEVD01000030.72.1605 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;Streptococcus infantis ATCC 700779 87.7 1551 -1 91.0 1538 1490 61 130 1360
\ntongue.PFemirge_126 0.011402 FJ470572.1.1411 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;[Eubacterium] oxidoreducens group;uncultured bacterium 96.5 1362 -1 96.8 1358 1354 8 40 1314
\ntongue.PFemirge_675 0.011261 AEVD01000030.72.1605 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;Streptococcus infantis ATCC 700779 86.8 1553 -1 90.2 1538 1488 65 140 1348
\ntongue.PFemirge_1236 0.011060 FM872752.1.1502 Bacteria;Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Granulicatella;uncultured bacterium 99.9 1458 -1 99.8 1461 1458 0 1 1457
\ntongue.PFemirge_285 0.011031 GQ338720.1.1529 Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonella;uncultured bacterium 98.4 1529 -1 98.4 1529 1529 0 25 1504
\ntongue.PFemirge_764 0.010882 ACSB01000014.3447.4963 Bacteria;Actinobacteria;Actinobacteria;Micrococcales;Micrococcaceae;Rothia;Rothia mucilaginosa M508 99.4 1451 -1 99.7 1451 1444 7 2 1442
\ntongue.PFemirge_585 0.009756 AENQ01000044.3766.5256 Bacteria;Proteobacteria;Epsilonproteobacteria;Campylobacterales;Campylobacteraceae;Campylobacter;Campylobacter concisus UNSWCD 99.9 1491 -1 99.9 1491 1491 0 1 1490
\ntongue.PFemirge_404 0.007731 KC169767.1.1488 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;uncultured Bacteroidetes bacterium
\n99.2 1487 -1 99.2 1487 1487 0 12 1475
\ntongue.PFemirge_100 0.006757 EU531780.1.1546 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured bacterium 99.2 1528 -1 99.2 1528 1528 0 12 1516
\ntongue.PFemirge_101 0.006259 FJ558479.1.1379 Bacteria;Actinobacteria;Actinobacteria;Micrococcales;Micrococcaceae;Rothia;uncultured bacterium 96.6 1388 -1 97.6 1379 1370 18 29 1341
\ntongue.PFemirge_303 0.005992 AY923125.1.1558 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;Streptococcus sp. oral clone ASCB12 98.0 1548 -1 98.1 1544 1540 8 23 1517
\ntongue.PFemirge_259 0.005546 AY349396.1.1524 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 6;Prevotella sp. oral clone GI032 99.3 1509 -1 99.2 1509 1509 0 11 1498
\ntongue.PFemirge_281 0.005305 JN379060.1.1513 Bacteria;Firmicutes;Bacilli;Bacillales;Family XI;Gemella;uncultured bacterium 97.6 1512 -1 97.5 1512 1512 0 37 1475
\ntongue.PFemirge_613 0.004982 AWVR01000095.17.1514 Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;Leptotrichia sp. oral taxon 215 str. W9775 99.2 1415 -1 99.2 1415 1413 2 9 1404
\ntongue.PFemirge_488 0.004680 AB637220.1.1505 Bacteria;Firmicutes;Bacilli;Bacillales;Planococcaceae;Planomicrobium;uncultured bacterium 92.0 1507 -1 92.0 1505 1503 4 117 1386
\ntongue.PFemirge_718 0.004675 FJ470510.1.1487 Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales;Flavobacteriaceae;Capnocytophaga;uncultured bacterium 88.9 1492 -1 89.2 1487 1482 10 155 1327
\ntongue.PFemirge_1 0.004483 HQ778352.1.1429 Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Fusobacteriaceae;Fusobacterium;uncultured organism 98.3 1383 -1 98.2 1383 1382 1 23 1359
\ntongue.PFemirge_138 0.004463 JQ471598.1.1397 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 7;uncultured bacterium 99.3 1395 -1 99.2 1395 1395 0 10 1385
\ntongue.PFemirge_4 0.004402 JQ460226.1.1424 Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonella;uncultured bacterium 99.4 1424 -1 99.4 1424 1423 1 7 1416
\ntongue.PFemirge_390 0.004301 HK241664.1.1506 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Alloprevotella;unidentified 97.8 1505 -1 97.7 1505 1505 0 33 1472
\ntongue.PFemirge_331 0.004169 JQ449434.1.1369 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Peptostreptococcus;uncultured bacterium 99.5 1368 -1 99.3 1368 1367 1 6 1361
\ntongue.PFemirge_190 0.004099 AF385510.1.1522 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Stomatobaculum;Eubacterium sp. oral clone DO016 98.5 1510 -1 98.5 1510 1509 1 22 1487
\ntongue.PFemirge_653 0.004050 AM420032.1.1494 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella 7;uncultured Prevotella sp. 99.0 1493 -1 98.9 1492 1491 2 13 1478
\ntongue.PFemirge_407 0.004045 JQ460279.1.1390 Bacteria;Actinobacteria;Actinobacteria;Micrococcales;Micrococcaceae;Rothia;uncultured bacterium 74.5 1337 -1 79.6 1307 1265 72 269 996
\ntongue.PFemirge_747 0.003609 GU361882.1.1515 Bacteria;Firmicutes;Bacilli;Bacillales;Family XI;Gemella;uncultured bacterium 98.2 1515 -1 98.2 1515 1515 0 28 1487
\ntongue.PFemirge_41 0.003531 HQ771317.1.1450 Bacteria;Proteobacteria;Betaproteobacteria;Neisseriales;Neisseriaceae;Neisseria;uncultured organism 92.2 1315 -1 93.7 1311 1296 19 84 1212
\ntongue.PFemirge_2433 0.003313 GQ091776.1.1369 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured bacterium 97.7 1367 -1 97.7 1366 1365 2 29 1336
\ntongue.PFemirge_287 0.003290 FJ558066.1.1408 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured bacterium 97.5 1408 -1 97.5 1408 1408 0 35 1373
\ntongue.PFemirge_2259 0.003137 FJ470581.1.1490 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Lachnospiraceae UCG-008;uncultured bacterium 99.4 1490 -1 99.4 1490 1490 0 9 1481
\ntongue.PFemirge_44 0.003105 AWSC01000005.3443.4969 Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;Actinomyces graevenitzii F0530 99.7 1519 -1 99.7 1519 1519 0 4 1515
\ntongue.PFemirge_447 0.003031 FJ983043.1.1537 Bacteria;Firmicutes;Clostridia;Clostridiales;Peptostreptococcaceae;Peptoclostridium;uncultured bacterium 90.3 1530 -1 90.9 1523 1516 14 134 1382
\ntongue.PFemirge_220 0.003019 FJ470573.1.1497 Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Selenomonas;uncultured bacterium 99.1 1492 -1 99.0 1491 1490 2 12 1478
\ntongue.PFemirge_9891 0.002888 EU531782.1.1545 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured bacterium 78.6 1557 -1 82.2 1538 1485 72 261 1224
\ntongue.PFemirge_477 0.002708 HQ799343.1.1445 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Lachnoanaerobaculum;uncultured organism 96.0 1438 -1 96.5 1431 1425 13 45 1380
\ntongue.PFemirge_381 0.002644 HE681253.1.1518 Bacteria;Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcaceae UCG-014;uncultured bacterium 99.5 1508 -1 99.4 1508 1508 0 8 1500
\ntongue.PFemirge_195 0.002638 KF092280.1.1374 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured bacterium 93.9 1162 -1 94.8 1161 1158 4 67 1091
\ntongue.PFemirge_671 0.002544 AJ006963.1.1453 Bacteria;Firmicutes;Clostridia;Clostridiales;Family XIII;[Eubacterium] nodatum group;[Eubacterium] sulci 97.7 1453 -1 97.6 1491 1453 0 34 1419
\ntongue.PFemirge_1497 0.002483 JF189706.1.1349 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Oribacterium;uncultured bacterium 97.6 1344 -1 97.5 1344 1344 0 32 1312
\ntongue.PFemirge_813 0.002332 KF101495.1.1358 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Porphyromonadaceae;Porphyromonas;uncultured bacterium 82.5 1331 -1 83.9 1323 1308 23 210 1098
\ntongue.PFemirge_779 0.002305 EF404014.1.1511 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured bacterium 96.6 1416 -1 97.3 1416 1406 10 38 1368
\ntongue.PFemirge_9890 0.002145 HQ778326.1.1469 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;uncultured organism 95.0 1467 -1 95.6 1461 1455 12 61 1394
\ntongue.PFemirge_1246 0.002121 HQ805205.1.1478 Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Veillonella;uncultured organism 96.0 1479 -1 96.0 1478 1477 2 57 1420
\ntongue.PFemirge_339 0.002115 FJ557635.1.1415 Bacteria;Firmicutes;Negativicutes;Selenomonadales;Veillonellaceae;Megasphaera;uncultured bacterium 96.7 1415 -1 96.7 1415 1415 0 46 1369
\ntongue.PFemirge_147 0.002077 KF101349.1.1358 Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales;Prevotellaceae;Prevotella;uncultured bacterium 98.9 1358 -1 98.9 1358 1358 0 15 1343
\ntongue.PFemirge_247 0.001999 AF432138.1.1487 Bacteria;Fusobacteria;Fusobacteriia;Fusobacteriales;Leptotrichiaceae;Leptotrichia;Leptotrichia sp. oral clone FP036 99.1 1485 -1 99.1 1485 1485 0 13 1472
\ntongue.PFemirge_1087 0.001934 JF189684.1.1343 Bacteria;Actinobacteria;Actinobacteria;Micrococcales;Micrococcaceae;Rothia;uncultured bacterium 96.8 1347 -1 97.1 1343 1339 8 35 1304
\ntongue.PFemirge_9889 0.001919 AEVD01000030.72.1605 Bacteria;Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus;Streptococcus infantis ATCC 700779 84.6 1557 -1 88.4 1538 1484 73 167 1317
\ntongue.PFemirge_95 0.001868 FJ557743.1.1389 Bacteria;Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Stomatobaculum;uncultured bacterium 97.2 1368 -1 97.2 1368 1368 0 38 1330
\ntongue.PFemirge_235 0.001708 FJ470591.1.1499 Bacteria;Actinobacteria;Actinobacteria;Actinomycetales;Actinomycetaceae;Actinomyces;uncultured bacterium 98[\/vc_column_text][\/toggle][\/toggles][vc_column_text]PhyloFlash is going to try to reconstruct the taxonomic RNA subunits using three different methods. First by mapping the reads against a curated 16S rRNA SILVA<\/a> database, then using the mapped reads it will try to reconstruct the sequences using SPAdes<\/a> and EMIRGE<\/a>.<\/p>\n

From the previous file we can see that phyloFlash predicts around 200 OTUs in total. It also try to sort out the different taxa per abundance, based on the concept that the more a taxa is present in a sample the more sequenced reads it will be.<\/p>\n[\/vc_column_text][vc_row_inner text_align=”left”][vc_column_inner column_padding=”no-extra-padding” column_padding_position=”all” background_color_opacity=”1″ width=”1\/2″][vc_column_text]We know now that we should expect large diversity. From the different prediction methods it seems that even if the precise taxonomic identification varies,\u00a0 Betaproteobacteria, Bacterioidetes, Firmicutes and Gammaproteobacteria are among the most abundant taxa.<\/p>\n

On the right is a pie chart representing the class of different OTU predicted by the EMIRGE module. I only displayed the taxa predicted with a ratio above 0,5% of the total reads used. This is certainly biased but it help predicting what is in our library.[\/vc_column_text][\/vc_column_inner][vc_column_inner column_padding=”no-extra-padding” column_padding_position=”all” background_color_opacity=”1″ width=”1\/2″][vc_column_text]\n