Metagenoms

Web sites

• NCBI Bacterial Genomes 647 Complete, 1067 in Progress
• JGI Fidelity of Analysis of Metagenomic Samples (FAMeS)
• JGI Simultaed High Complexity (SIMHC)
• Human Microbiome Project (HMP) @ NIH
• WUSTL Human Gut Microbiome (HGMI) 41 genomes
• Baylor HMP list of 353 genomes targeted for sequencing by the 4 centers
• JCVI HMP 50 genomes (oral,skin,vagina)
• JGI IMG
• HGMI at NIH
• HGMI TA ~150K Sanger traces; trimming points are given

Articles

• Kurokawa
• Gill
• JGI
• HMP Nature Oct 2007
• Human Microbiome Pilot Project (HMPP)
• Human Gut Microbiome Initiative (HGMI) Need for more RefSeqs; sequence the genomes of 100 cultured representatives of the phylogenetic diversity in the human gut microbiota
• Nature Reviews: Microbiology in the post-genomic era
• IMG NAR 2007
• Accurate phylogenetic classification of variable-length DNA fragments., Nature Methods Jan 2007

Lineage

 domain  
 phylum  
 class   
 order   
 family  
 genus   
 species 
 strain

Example: Pseudomonas aeruginosa

 domain: Bacteria
 phylum: Proteobacteria
 class:  Gammaproteobacteria
 order:  Pseudomonadales 
 family: Pseudomonadaceae
 genus:  Pseudomonas
 species:Pseudomonas aeruginosa group
 strain: Pseudomonas aeruginosa

HMP

NIH Roadmap

1. Sequence the genomes of 200 microbes that have been isolated from the human body;
2. Recruit a set of healthy donors and obtain samples from a set of body regions
3. Perform initial 16S rDNA gene metagenomic sequence analyses to estimate the complexity of the microbiota at these sites.

Centers: Baylor, Broad, JCVI, WUSTL

• ~10times more bateria cells than human cells in the body
• small-subunit (16S) ribosomal RNA gene-sequence-based surveys:

 * found in all microorganisms 
 * has enough sequence conservation for accurate alignment 
 * has enough variation for phylogenetic analyses.

• skin, mouth, oesophagus, stomach, colon and vagina
• largest reported data sets are for the gut
• most of the 10–100 trillion microorganisms in the human gastrointestinal tract live in the colon.
• more than 90% of all phylogenetic types (phylotypes) of colonic bacteria belong to just 2 of the 70 known divisions (phyla) in the domain Bacteria: the Firmicutes and the Bacteroidetes.

Firmicutes (Gram-positive bacteria) : 639 Genome Sequences

   * Bacilli    472
   * Clostridia    106
   * Erysipelotrichi    1
   * Mollicutes    60
   * Thermolithobacteria   
   * unclassified Firmicutes sensu stricto   
   * environmental samples    

Bacteroidetes : 53 Genome Sequences

   * Bacteroidetes (class)    22
   * Flavobacteria    21
   * Sphingobacteria    8
   * unclassified Bacteroidetes    1
   * environmental samples    1

Actinobacteria

• In colon, the differences between individuals are greater than the differences between different sampling sites in one individuall
• Comunirties are usually stable over time

SIMHC

Data

Online

• 113 reference genomes : 89 complete, 24 incomplete

                   #elem   min     max     mean    median  n50     sum
 NC_chromosomes    103     943016  8264687 3569251 3481691 4326849 367,632,882
 NC_plasmids       70      3361    821788  147207  96488   300758  10,304,479
 NC_*              173     3361    8264687 2184609 1966858 4317977 377,937,361   # come from 89 genomes & 70 plasmids ; some genomes contain multiple chromosomes

 NZ_*              3505    185     1802798 26389   9051    73891   92,494,763    # come from 24 genomes

 N*_total          3678    185     8264687 127904  9940    3561584 470,432,124

• 118084 Sanger reads from 3 insert libs (small,med,large)

 #reads  min     max     mean    median  n50     sum
 116771  43      3754    950     968     982     110875383

• More than 50% of reads are from Proteobacteria; Gammaproteobacteria

 Proteobacteria    73261
 Firmicutes        15707
 Actinobacteria    10744
 ...

• Organisms

 Burkholderia      12431 # Which one? B. cenocepacia AU 1054 , B. AMMD, B. sp. 383, B. xenovorans LB400, B. cenocepacia HI2424, B. vietnamiensis G4
 Shewanella        9613
 Rhodopseudomonas  5279
 ...
 Ferroplasma       471
 Pediococcus       456
 Oenococcus        422    # Firmicutes, Bacilli, Lactobacillales

• read coverage of each organism 0.09 ..0.53X

 0.53X: Moorella thermoacetica ATCC 39073 (1426 reads; NC_007644 2,628,784bp 55.79%GC) (Firmicutes)
 0.09X: Xylella fastidiosa Ann-1

Local

• 113 reference genomes : 89 complete, 24 incomplete
• 116771 Sanger reads from 3 insert libs (small,med,large)
• 118084-116771=1313 Moorella and Xylella were discarded !!!
• read coverage of each organism ...

 0.23X: Moorella thermoacetica ATCC 39073 (740 reads; NC_007644 2,628,784bp 55.79%GC) (Firmicutes)

Assemblies

Contig stats

                      #elem   min     max     mean    median  n50     sum             singl

 phrap-ctg            23398   73      8603    1289    1194    1341    30,163,430      66524
 arachne-ctg          578     240     6300    1878    1822    1985    1,085,508       115300
 jazz-scaff           860     1000    39837   3236    1105    7278    2,783,247       109080

 CA-scaff             4327    1000    48861   1682    1342    1456    7,278,044       76270     # OBT trimming of the reads
 CA-scf-unambiguous   4327    1000    26070   1473    1342    1403    6,374,650
 CA-ctg               4491    1000    5252    1419    1340    1388    6,374,863                 # 4407 CONTAINED in references
 CA-deg               11611   66      5920    850     833     898     9,865,902

 AMOS-ctg             95372   72      7426    975     916     964     92,959,055      5661      # alignment based trimming of the reads; casm-layout "-S -r" have been used
 AMOS-ctg-plasmids    2078    159     7426    1007    911     972     2,092,115

 minimus2-ctg         15211   85      4699    1257    1205    1305    19,119,413      82535     # alignment based trimming of the reads

CA:

• Longest scaff: scf7180000043951 48861bp(26070 unambiguous) comes from NC_007968 41221 38.26 Psychrobacter cryohalolentis K5 plasmid 1
• Longest ctg: ctg7180000029682 5252bp comes from NC_008499 35595 38.51 Lactobacillus brevis ATCC 367 plasmid 2
• Longest degen: ctg7180000030915 5920bp NC_008608 30722 56.32 Pelobacter propionicus DSM 2379 plasmid pPRO2

minimus2 on AMOScmp & CA (ctg+degen) : most of CA assemblies were contained in AMOScmp contigs; stats about same as AMOScmp