Bacillus anthracis

Background

• 89 known strains
• Most virulent: Ames(USA 2001), Vollum (WWII, biological weapon)
• Benign: Sterne (used as vaccine)

Virulence factors that distinguish Bacillus anthracis from Bacillus cereus are encoded on two plasmids, pXO1 (anthrax toxin) and pXO2 (capsule genes). The capsule protects against phagocytosis once the vegetative bacterium enters the bloodstream. The anthrax toxin consists of 3 components, a protective antigen (PA), lethal factor (LF), and edema factor (EF). PA/LF and PA/EF complexes are internalized by host cells where the LF (metalloprotease) and EF (calmodulin-dependent adenylate cyclase) components act. At high levels LF induces cell death and release of the bacterium while EF increases host susceptibility to infection and promotes fluid accumulation in the cells. (NCBI)

• Ames, AmesAncestor, Stern are 99.9% identical; no rearrangements
• The chromosome and plasmids don't seem to share sequence; few < 2Kb alignments at < 92%id

• TIGR Publication

Genome Projects (listed by NCBI)

NIH Genome Projects

 Center          Complete     Assembly    Progress  Total
 TIGR/JCVI       2            8           1         11
 LANL            0            6           0         6
 DOE             1            0           0         1 
 NMRC            0            0           2         2
 Total           3            14          3         20

TIGR/JCVI Strains

 Contigs  Traces   Status    Completed   Strain 
 0        96,532   Progress  .           A0039  
 62       67,600   Assembly  2007/07/25  Tsiankovskii-I AA ; ??? possible update
 1(+2)    101,379  Complete  2004/05/20  Ames Ancestor (Ames 0581)  AA ; Insignia; pXO1, pXO2
 42       86,181   Assembly  2004/06/04  A1055 AA ; Insignia
 1(+2)469 83,552   Assembly  2005/05/16  A2012  Insignia; The  1 contig contains 469 gaps; 65508 of the traces have no qualities; pXO1, pXO2
 1        125,879  Complete  2002/05/16  Ames  ??? complete but not in AA ; Insignia
 49       0        Assembly  2004/06/07  Australia 94   ??? no TRACES ; Insignia
 30       90,308   Assembly  2004/06/04  CNEVA-9066 Insignia
 64       92,429   Assembly  2004/06/07  Kruger B AA ; Insignia
 52       103,144  Assembly  2004/06/04  Vollum Insignia
 44       95,078   Assembly  2004/06/07  Western North America USA6153 Insignia

LANL Strains

No traces in TA; none in Insignia

 Contigs  Traces   Status    Completed    Strain       
 60       0        Assembly  2008/04/08   A0174
 60       0        Assembly  2008/02/12   A0193                              
 68       0        Assembly  2008/03/24   A0389
 46       0        Assembly  2008/02/12   A0442
 57       0        Assembly  2008/03/24   A0465
 63       0        Assembly  2008/01/16   A0488

+ 2 plasmid genome projects (pX01,pX02) completed in 1999

DOE Strains

 Contigs  Traces   Status    Completed    Strain
 1        147,665  Complete  2004/06/24   Sterne ??? complete but not in AA; Insignia; pXO1, pXO2

Naval Medical Research Center

 Contigs  Traces   Status    Strain
 0        0        Progress  34F2(NMRC)
 0        0        Progress  34F2 delta gerH

Complete

 Strain        Status       chromosome      pXO1       pXO2
 A2012         Assembly     .               181677     94829 
 Ames          Complete     5227293         .          .
 AmesAncestor  Complete     5227419         181677     94830
 Sterne        Complete     5228663         181654     96231

Genome Projects (not listed by NCBI)

BCM

Data available "by request"

 Strains  reads    cvg   ctgs   N50ctg    AssemblyDate
 31-101   451,308  9.4   3,418  2,592     5-22-2006
 500      363,269  7.6   5,578  1,744     5-22-2006

Strain Assemblies

A2012

• NCBI Genome

              RefId           Len     GC%
 chromosome   NZ_AAAC02000001 5093554 35.36
 pXO1         NC_003980       181677  32             # 100%   identical to AmesAncestor pXO1
 pXO2         NC_003981       94829   33             # 99.99% identical to AmesAncestor pXO2; 1 del

 chromosome
       #elem   min     max     mean    median  n50     sum
 ctg   425     94      132589  11885   6855    24366   5051252 # 42,346 N's

Traces:

• 18,045 reads have qual. & 65,507 don't

 Libraries:
 Lib             Mean    Stdev           Count
 T13322          2000    600             32133
 T13323          4000    1200            31455
 1047127226559   2000    600             18036
 T10914          3000    900             1719
 T10930          10000   3000            150
 GBZH            4500    .               29
 ...
 Total                                   83553

CA

Summary

         #elem   min     max     mean    median  n50     sum
 scf     325     1001    245405  16824   1250    86284   5467834
 ctg     476     844     122753  11450   2025    33522   5450075   # larger N50 than the NCBI assembly
 deg     1760    172     4878    852     818     882     1498894

0cvg(no plasmids)

         #elem   min     max     mean    median  n50     sum
 1con    223     1       783     91.28   33      252     20355
 ctg-deg 973     1       10316   239     140     444     232946

0cvg(including plasmids)

         #elem   min     max     mean    median  n50     sum
 ctg-deg 841     1       3183    183     82      402     154114

!!! there are some regions in the CA assembly not present in the reference

CA bog

         #elem   min     max     mean    median  n50     sum
 scf     334     930     282449  16611   1227    92676   5547969
 ctg     513     643     105647  10776   1630    36335   5528273
 deg     1898    172     8320    875     827     883     1659854

bog vs unitigger:

        count avg  max  N50  totalBases
scaff    +    -    +    +    +
ctg      +    -    -    +    +
deg      +    +    +    .    +
sur      +    -    +    .    +
utg      -    +    +    .    .
sing     -    .    .    .    .

AMOScmp-alignmentTrimmed

• Ref: Ames

       #elem   min     max     mean    median  n50     sum
 ctg   123     122     215418  42281   24322   98944   5200569

!!! larger contigs than NCBI/CA assembly

• Ref : A2012 pXO1 => 1ctg
• Ref : A2012 pXO2 => 1ctg & 2 snps

Ames

• Complete

 NC_003997.3    5227293 35.38  Bacillus anthracis str. Ames, complete genome

• not in AA

AMOScmp-alignmentTrimmed

• no 0 cvg regions when factory trimmed reads aligned to it
• -D LAYERR=90 => 1 piece

 ref=5227293 bp
 assembly=5227311
 amosvalidate=>1555 snps
 nucmer align of assembly to ref & filter -q => 287 snps

• Many stretched & missoriented mates
• Location: /fs/szasmg3/dpuiu/Bacilus_anthracis/Ames/Assembly/2008_0820_AMOScmp-alignmentTrimmed

CA

Output:

         #elem   min     max     mean    median  n50     sum       snps
 scf     59      1021    893362  83396   1970    593436  4920387
 ctg     67      1021    736364  73430   2483    280568  4919826   496
 deg     245     70      141988  2016    671     34515   493924

• There are many stretched mates; no compressed ones !!!

0cvg

         #elem   min     max     mean    median  n50     sum
 1con    10      9       804     147     80      804     1467
 ctg-deg 224     1       2197    604     633     715     135220

Ctg 0 cvg regions:

 #id                   len     gc%     start   end     len     cvg
 ctg7180000001099      2637    35.04   439     2637    2198    0        33.97% gc ; blastn:96% identity to Bacilus cereus ; blastx: alpha/beta hydrolase & protein disulfide isomerase hits
 ctg7180000001288      28653   36.17   19125   19233   108     0        52.29% gc ; blastn: 100% identity to Ames Ancestor & Sterne
 ctg7180000001288      28653   36.17   28636   28653   17      0        aligned to other Bacillus anthracis strains
 ctg7180000001300      156978  35.35   1       161     160     0        46.58% gc ; clonning vector
 ctg7180000001302      70072   34.28   69464   70072   608     0        55.83% gc ; clonning vector

Deg 0 cvg regions:

 all have high GC% (>48.24%) probably cloning vector

Ref breaks:

 NC_003997.3   145564
 NC_003997.3   627742
 NC_003997.3   1151234
 NC_003997.3   2085561
 NC_003997.3   3515380

CA bog

         #elem   min     max     mean    median  n50     sum
 scf     94      1006    1158003 55534   14912   636683  5220223
 ctg     103     1006    594903  50630   15054   280568  5214932
 deg     501     70      15945   1044    609     1432    522839
 

0cvg

         #elem   min     max     mean    median  n50     sum
 1con    7       9       120     72.14   75      112     505
 ctg-deg 236     3       2197    607     632     713     143262

Ref breaks:

 NC_003997.3     145564
 NC_003997.3     627742
 NC_003997.3     1151234
 NC_003997.3     2085561
 NC_003997.3     3515380

bog vs unitigger:

        count avg  max  N50  totalBases
scaff    +    -    +    -    +
ctg      +    -    -    +    +
deg      +    -    -    .    +
sur      +    -    -    .    +
utg      +    -    +    .    .
sing     -    .    .    .    .

Ames Ancestor

• Complete & in AA

 NC_007530.2    5227419 35.38  Bacillus anthracis str. 'Ames Ancestor', complete genome
 NC_007322.2    181677 32.53  Bacillus anthracis str. 'Ames Ancestor' plasmid pXO1, complete sequence
 NC_007323.3    94830  33.04  Bacillus anthracis str. 'Ames Ancestor' plasmid pXO2, complete sequence
 total          5503926

• Downloaded from AA and converted to bank
• Location: /fs/szasmg3/dpuiu/Bacilus_anthracis/AmesAncestor/CS--AI-293

CA

         #elem   min     max     mean    median  n50     sum
 ctg     13      1316    2750171 413057  187947  2750171 5369744
 deg     11      271     62530   13876   1627    62530   152635
 ctg+deg 24      271     2750171 230099  37452   2750171 5522379
 scf     11      1316    2750171 488185  212112  2750171 5370036

Ref: no alignment breaks, no 0cvg regions

Sterne

• Complete but not in AA.

            RefId        Len        GC%
 chromosome NC_005945.1  5228663   35.38
 pXO1       NC_001496.1  181654    32
 pXO2       NC_002146.1  96231     33 

!!! the plasmids are not listed with the genome project

• Traces available in TA. Looks like some reads are missing; I'm getting many 0 cvg regions when aligning the reads to the finished genome

       #elem   min     max     mean    median  n50     sum
 0cvg  53      8       4946    529     272     1508    28017

AMOScmp

• version: June 12 2007
• untrimmed reads => 358 ctg
• 53 zero cvg regions, max is almost 5K

AMOScmp-alignmentTrimmed

• reads are trimmed according to alignment coords

       #elem   min     max     mean    median  n50     sum
 ctg   46      736     468060  112792  49000   355370  5188425

CA

• runCA-OBT.pl script
• version 5.1
• Output:

 #elem   min     max     mean    median  n50     sum
 ctg     204     1000    468299  26560   1310    189443  5418252
 deg     145     266     32820   1756    804     21949   254596
 ctg+deg 349     266     468299  16255   1146    181331  5672848
 scf     186     1000    671877  29170   1274    294809  5425585
 singleton  2418

Ctg 0 cvg regions:

 all have high GC% (>52.08%) probably cloning vector

Deg 0 cvg regions: actually they align to pXO1

 #id                             len     gc%
 deg7180000001258.1-297          297     31.65
 deg7180000001300.420-6468       6049    32.05
 deg7180000001254.1-28442        28442   33.01
 deg7180000001258.557-1060       504     34.52
 deg7180000001253.1-576          576     35.76
 deg7180000001300.6728-6824      97      36.08
 deg7180000001300.1-160          160     38.12
 ..

minimus2

Input:

                 #elem   min     max     mean    median  n50     sum
 AMOS            46      736     468060  112792  49000   355370  5188425
 CA              349     266     468299  16255   1146    181331  5672848
 AMOS+CA         395     266     468299  27497   1210    215336  10861273

Output:

                 #elem   min     max     mean    median  n50     sum
 ctg             47      931     468316  110527  37238   355370  5194753
 singl           282     24      32820   1602    1053    1341    451821
 ctg+singl       329     24      468316  17163   1103    296377  5646574

Vollum (DOE)

NCBI data:

         #elem   min     max     mean    median  n50     sum
 ctg     52      311     812727  105547  29178   400992  5488459

CA

         #elem   min     max     mean    median  n50     sum
 ctg     39      1073    1541457 136843  51912   422289  5336858
 deg     18      693     54711   7994    2244    54711   143898
 scf     25      1440    1593252 214001  94958   676360  5350037

!!! Bigger contigs

• No alignment breaks vs NCBI assembly

CA bog

Same number of scf & ctg; fewer & longer unitigs than CA

A0442 (LANL)

• no traces
• NCBI

       #elem   min     max     mean    median  n50     sum
 ctg   46      7229    1040654 116844  74960   223192  5374836

Some contigs align to Ames Ancestor pXO1 & pXO2 There are some "unique" regions in A0442 not present in Ames Ancestor