Pseudodomonas syringae: Difference between revisions

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   NC_004632.1    67,473    56.17
   NC_004632.1    67,473    56.17


Little similarity between the chromosome and plasmids.
The 2 plasmids share a significant amount of DNA; see
The 2 plasmids share a significant amount of DNA; see
/fs/szasmg2/Bacteria/Pseudomonas_syringae/Data/nucmer/NC_004633-NC_004632.png
  /fs/szasmg2/Bacteria/Pseudomonas_syringae/Data/nucmer/NC_004633-NC_004632.png


=== UNC: Jeff Dangl ===
=== UNC: Jeff Dangl ===

Revision as of 19:16, 14 February 2008

Pseudomonas syringae pv. tomato str. DC3000

Originally sequenced and finished at TIGR: published Sept 2003

Data

NCBI

 AA: no assembly
 TA 80,959 reads 
 Genome Project
 Taxonomy TaxId=223283

Chromosome + 2 plasmids:

 Name           Length    %GC
 NC_004578.1    6,397,126 58.40
 NC_004633.1    73,661    55.15
 NC_004632.1    67,473    56.17

Little similarity between the chromosome and plasmids. The 2 plasmids share a significant amount of DNA; see

 /fs/szasmg2/Bacteria/Pseudomonas_syringae/Data/nucmer/NC_004633-NC_004632.png

UNC: Jeff Dangl

New sequence:

 * Solexa 3 lanes; 
 * 454 shotgun 1/4 Plate (250bp read); 
 * 454 paired ends 1/4 Plate : 
     * contain a 44 bp linker in the middle
     * the linker sequence is: GTTGGAACCGAAAGGGTTTGAATTCAAACCCTTTCGGTTCCAAC
     * there are some (not many) 454 paired end sequences that contain multiple instances of the linker (tandem): Example EUEIEUN01ANUGL_length=128_xy=0154_1891 

Data stats

 .                               #elem             min     median  max     sum             mean    stdev   n50
 DC3000.reads.filtered.fasta     6,340,136         32      32      32      202884352       32      0       32      DC3000 Solexa Reads
 DC3000.format.454Reads.fna      123,992           38      86      329     15623908        126.01  58.89   142     DC3000 454 Paired Reads (forward+linkerr+reverse)
 DC3000.TCA.454reads.format.fna  77,466            35      244     371     18627363        240.46  26.85   245     DC3000 454 Reads
 DC3000Plasmids.fa               2                 67473   73661   73661   141134          70567   3094    73661   Pseudomonas syringae pv. tomato DC3000 Plasmids
 Psudomonas_syringae.fa          1                 6397126 6397126 6397126 6397126         6397126 0       6397126 Pseudomonas syringae pv. tomato DC3000 reference
 
 
 Quality values are missing for all data sets!!!
 I assigned default qual=3 to all the base (.frg & .afg files)  

UNC sequence data: (not avail any more?)

 http://biology622.dhcp.unc.edu/~labweb/DCData/

UNC (e-mail):

 * Theoretical minimum number of contigs we can obtain is 268 (our reads fail to cover 269 nucleotides). 
 * Our de novo assembly spans the genome in 853 contigs totaling 6,313,026 bp. 
 * 98.7% of the genome is covered by a contig; 
 * 84% of the genome is covered by contigs 10,000 bp or greater. 
 * The average gap size between contigs is 98 bp; 
 * average contig size 7401 bp. 
 * The N50 = 37,444 bp. 
 * Our largest BAMBUS "scaffold" is 2,565,761 bp

Files location:

 /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Data
 /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Assembly

Assemblies

CBCB (old)

!!! All AMOSCmp assemblies contain tandem duplications in Solexa only coverage areas

1. AMOSCmp

 454 single reads + Solexa reads 
 /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Assembly/Solexa-454/2007_1009_AMOSCmp-relaxed
 142 contigs (37 negative gaps, 89 positive gaps)
 No read trimming was done. 
 AMOScmp used the following parameters:
   nucmer -c  20
   casm-layout -t 20 -o 5
 "-t 20" allows for 20 bp long  dirty sequence ends which seem to solve the "low quality" problem.
 => 22 large contigs
 
 454 single reads + 30 bp Solexa reads  => 167 contigs , 49 negative gaps, 100 positive gaps 
 454 single reads + 25 bp Solexa reads  => 293 contigs,  144 negative gaps, 131 positive gaps 

2. AMOSCmp

 454 single reads + Solexa reads + 454 paired ends
 Only the 454 paired ends that contain 1 single complete adaptor sequence were used (allmost all)
 /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Assembly/Solexa-454-454p/2007_1011_AMOSCmp-relaxed-filtered
 149 contigs; very similar to the prev ome

3. AMOSCmp (MAJORITY=50) -> best

 454 single reads + Solexa reads 
 /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Assembly/Solexa-454/2007_1015_AMOSCmp-relaxed-MAJORITY50 
 131 contigs  (18 negative gaps)
 No read trimming was done. 
 AMOScmp used the following parameters:
   nucmer -c  20
   casm-layout -t 20 -o 5 -m 50
 No read trimming was done. 
 "-t 20" allows for 20 bp long  dirty sequence ends which seem to solve the "low quality" problem.
 "-m 20" merges some contigs together
 => 10 large contigs
 contig#        len     gc%
 4              2290968 59.00
 7              1817904 58.18
 3              1405326 58.08
 5              648413  58.48
 2              192413  57.86
 6              87152   58.02
 131            71251   56.47
 1              32939   54.86
 130            29120   59.36
 9              20309   53.56
 95             3589    59.46


 Rerun Solexa32,Solexa30,Solexa25 with "nucmer -b 2 -g 5"
 
 2007_1015_AMOSCmp-relaxed-Solexa32/
 2007_1015_AMOSCmp-relaxed-Solexa30/
 2007_1015_AMOSCmp-relaxed-Solexa25/
 /fs/szasmg2/Bacteria/Pseudomonas_syringae/Assembly/Solexa-454/qc.combine.3
 
 $ show-coords 1con-contigs.delta | grep gi | awk '{print $7}' | getSummary.pl # sum of ref alignments: 13608985
 $ show-coords 1con-contigs.delta | grep gi | awk '{print $8}' | getSummary.pl # sum of qry alignments: 13747738
  138,753 bp in duplications for Solexa32 ??? 
   61,741 bp in duplications for Solexa30 ??? 
   10,881 bp in duplications for Solexa25 ??? 
 Copy of assembly files:
  /fs/ftp-cbcb/pub/data/dpuiu/Pseudomonas_syringae
  ftp://ftp.cbcb.umd.edu/pub/data/dpuiu/Pseudomonas_syringae/Solexa-454

4. AMOSCmp

 Sanger reads
 /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Assembly/Sanger/2007_1011_AMOSCmp-relaxed
 Many miss-oriented mates in the 4.8M-5M region of the chromosome
 22 contigs
 Chromosome
 Chromosome problem

5. Celera 3.11

 Sanger reads
 /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Assembly/Sanger/2007_1011_WGA
 22 scaff, 46 contigs, 181 degens
 Scaffold 7180000001443 looks circular: possible 163,074 bp plasmid
 aligns to 4.8M-5M "problem" region in the chromosome
 7180000001443.png
     [S1]     [E1]  |     [S2]     [E2]  |  [LEN 1]  [LEN 2]  |  [% IDY]  |  [LEN R]  [LEN Q]  |  [COV R]  [COV Q]  | [TAGS]
 ===============================================================================================================================
        1   175592  |        1   175592  |   175592   175592  |   100.00  |   175592   175592  |   100.00   100.00  | 7180000001443   7180000001443   [IDENTITY]
        1    12519  |   163075   175592  |    12519    12518  |    99.98  |   175592   175592  |     7.13     7.13  | 7180000001443   7180000001443   [BEGIN]
   163075   175592  |        1    12519  |    12518    12519  |    99.98  |   175592   175592  |     7.13     7.13  | 7180000001443   7180000001443   [END]


     [S1]     [E1]  |     [S2]     [E2]  |  [LEN 1]  [LEN 2]  |  [% IDY]  |  [LEN R]  [LEN Q]  |  [COV R]  [COV Q] | [TAGS]
 ===============================================================================================================================
  4790727  4911492  |   120764        1  |   120766   120764  |    99.98  |  6397126   175592  |     1.89    68.78  | gi|28867243|ref|NC_004578.1|    7180000001443
  4898971  4955870  |   175592   118697  |    56900    56896  |    99.98  |  6397126   175592  |     0.89    32.40  | gi|28867243|ref|NC_004578.1|    7180000001443

6. AMOSCmp (Chromosome+3 plasmids ref)

 Sanger reads
 Reference=complete genome(chromosome+3 plasmids) use "circular contig" in Celera 3.11 assembly
 /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Assembly/Sanger/2007_1012_AMOSCmp-relaxed-3plasmids
 38 contigs: 15 for main chromosome, 1 for longer plasmid, 21 for shorter plasmid, 1 for "circular contig"
 The missoriented read pile corresponding to the chromosome (4. AMOSCmp of Sanger reads) has dissapeared
 AA ready for submission: /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Assembly/Sanger/2007_1012_AMOSCmp-relaxed-3plasmids/AA/umd-20071030-141700.tar.gz


Solexa assemblied for different read coverages

Location

 /fs/szasmg2/Bacteria/Pseudodomonas_syringae/Assembly/Solexa/sample/
 
 Several AMOScmp assemblies, using 100%, 90% ... 10% of the P. syringae Solexa reads. 
 These would correspond to 30X, 27X, 24X .. 3X coverage 
 The read sampling was done randomly. One sample set for each coverage.


 
 The contig sequences were generated using AMOS bank2fasta. EMBOSS infoseq was used to get contig lengths. 
 The positive gap sizes (bases not covered) were taken from the .scaff file.
 ~dpuiu/bin/getSummary.pl was used to compute contig/gap summaries(mean/max/sum ...)
 

Chromosome + 2 plasmids

qc stats for Solexa assemblies done at different coverage levels

 cvg: 30,27,24...3
   
 $ more contig.chromo.summary positiveGaps.chromo.summary
 ::::::::::::::
 contig.summary
 ::::::::::::::
 %reads  #elem   #elem0  #elem<0 min     median  max     sum     mean    stdev   n50
 100     5502    0       0       32      338     32148   7296600 1326.17 2157.6  3714
 90      6463    0       0       32      330     25252   7252304 1122.13 1799.43 3009
 80      7570    0       0       32      303     20690   7209479 952.38  1487.03 2573
 70      9030    0       0       32      309     26306   7170384 794.06  1219.53 1986
 60      10571   0       0       32      295     22249   7124996 674.01  961.22  1608
 50      12598   0       0       32      274     22204   7075934 561.67  767.55  1266
 40      15343   0       0       32      252     9176    7011485 456.98  575.64  934
 30      21248   0       0       32      202     7751    6931907 326.24  376.06  597
 20      38702   0       0       32      117     3276    6807914 175.91  178.92  278
 10      84545   0       0       32      56      2652    6267925 74.14   57.62   90
 ::::::::::::::
 positiveGaps.summary
 ::::::::::::::
 %reads  #elem   #elem0  #elem<0 min     median  max     sum     mean    stdev   n50
 100     117     10      0       0       22      3065    19625   167.74  418.75  1308
 90      130     16      0       0       19      2100    19725   151.73  369.07  1211
 80      142     18      0       0       15      2174    20034   141.08  361.86  1209
 70      178     15      0       0       9       3417    20443   114.85  395.13  1823
 60      263     35      0       0       6       3875    21161   80.46   345.97  1457
 50      450     64      0       0       4       3398    22305   49.57   278.39  1823
 40      1047    156     0       0       4       3398    26488   25.3    173.77  929
 30      2915    446     0       0       4       3426    39094   13.41   115.74  104
 20      11154   1324    0       0       5       3420    110485  9.91    57.22   19
 10      44751   3321    0       0       9       3875    631930  14.12   35.45   25

Chromosome (only)

 $ more contig.chromo.summary positiveGaps.chromo.summary
 ::::::::::::::
 contig.chromo.summary
 ::::::::::::::
 %reads  #elem   #elem0  #elem<0 min     median  max     sum     mean    stdev   n50
 100     5352    0       0       32      387     18942   7152892 1336.49 2069.25 3674
 90      6313    0       0       32      362     16470   7110882 1126.39 1721.34 2969
 80      7411    0       0       32      322     15227   7069778 953.96  1436.49 2521
 70      8865    0       0       32      324     14901   7032202 793.25  1154.9  1968
 60      10406   0       0       32      304     10231   6988498 671.58  919.5   1586
 50      12389   0       0       32      279     7246    6941706 560.31  733.75  1247
 40      15131   0       0       32      256     5409    6879810 454.68  554.17  920
 30      20998   0       0       32      204     4102    6801160 323.9   358.93  588
 20      38368   0       0       32      117     2220    6680303 174.11  170.14  274
 10      83839   0       0       32      56      762     6144687 73.29   51.16   89
 ::::::::::::::
 positiveGaps.chromo.summary
 ::::::::::::::
 .       #elem   #elem0  #elem<0 min     median  max     sum     mean    stdev   n50
 100     15      5       0       0       1       33      107     7.13    10.84   33
 90      24      7       0       0       2       42      146     6.08    10.38   42
 80      38      11      0       0       2       36      212     5.58    8.84    26
 70      76      11      0       0       3       33      413     5.43    6.66    11
 60      163     29      0       0       4       33      1016    6.23    7.04    13
 50      347     60      0       0       3       49      1843    5.31    6.45    11
 40      947     151     0       0       4       53      5709    6.03    7.18    12
 30      2819    442     0       0       4       63      17882   6.34    7.34    12
 20      11029   1320    0       0       5       610     88516   8.03    10.84   15
 10      44485   3313    0       0       9       197     606841  13.64   15.05   24

 
 Nucmer was used to align contigs to reference
 "~dpuiu/bin/getNucmerCoverage.pl -M 0" was used to identify the 0 cvg regions 
 

Chromosome + 2 plasmids

 Table.? Gap sizes in P. syringae main chromosome & 2 plasmids for different Solexa assemblies
 
 $ more Solexa.coords.0cvg.summary 
 %reads   #elem   #elem0  #elem<0 min     median  max     sum     mean    stdev   n50
 100     104     0       0       1       62      1179    15804   151.96  236.77  486
 90      108     0       0       1       54      1697    15896   147.19  261.28  486
 80      117     0       0       1       35      1697    16057   137.24  253.9   486
 70      151     0       0       1       17      1697    16240   107.55  230.46  490
 60      223     0       0       1       10      1189    16872   75.66   177.66  455
 50      371     0       0       1       6       1703    17841   48.09   155.85  445
 40      888     0       0       1       5       1703    21504   24.22   104.94  296
 30      2539    0       0       1       5       1697    33875   13.34   63.75   36
 20      10198   0       0       1       6       1709    104225  10.22   33.56   17
 10      42284   0       0       1       10      1711    619965  14.66   21.88   24

Chromosome (only)

 Table.? Gap sizes in P. syringae main chromosome for different Solexa assemblies
 
 $ more Solexa.coords.0cvg.chromo.summary
 %reads  #elem   #elem0  #elem<0 min     median  max     sum     mean    stdev   n50
 100     6       0       0       1       17      33      94      15.67   12.85   33
 90      11      0       0       1       6       42      132     12      13.02   42
 80      21      0       0       1       6       35      199     9.48    10.38   26
 70      54      0       0       1       4       33      367     6.8     7.19    14
 60      124     0       0       1       5       33      922     7.44    7.36    13
 50      269     0       0       1       4       49      1768    6.57    6.68    11
 40      780     0       0       1       5       53      5428    6.96    7.08    11
 30      2432    0       0       1       5       63      17447   7.17    7.19    12
 20      10078   0       0       1       6       150     87195   8.65    8.97    14
 10      42115   0       0       1       10      197     601641  14.29   14.7    24
 => six 0 cvg regions in the chromosome if 100% of Solexa reads are used

Regions:

 Ref                             start   end
 gi|28867243|ref|NC_004578.1|    1022626 1022643 0
 gi|28867243|ref|NC_004578.1|    1206959 1206992 0 # near a transposease
 gi|28867243|ref|NC_004578.1|    3000373 3000405 0
 gi|28867243|ref|NC_004578.1|    3402234 3402240 0
 gi|28867243|ref|NC_004578.1|    3496311 3496312 0
 gi|28867243|ref|NC_004578.1|    4711568 4711573 0
 $ extractseq chromo.1con -regions '1022626-1022643,1206959-1206992,3000373-3000405,3402234-3402240,3496311-3496312,4711568-4711573' stdout -separate | awk '{print $1}'
 >NC_004578.1_1022626_1022643
 GGGGTTTTTATTGGGGCT
 
 >NC_004578.1_1206959_1206992  # near a transposease
 TAGAGATATTTTCAATACTAAAAAATATATTTTC
 
 >NC_004578.1_3000373_3000405
 GGCGCGACAGGCTTCCAGACGAGGTCTGCACGC
 
 >NC_004578.1_3402234_3402240
 CGGCTAC
 
 >NC_004578.1_3496311_3496312 
 GA
 
 >NC_004578.1_4711568_4711573 
 TGCCCG


CBCB (new)

Alignment based trimming

!!! Reduced the duplications significantly

Solution:

1. align all reads (Solexa) to the reference using nucmer. I initially used minmatch=20, mincluster=20 (-c 20 -l 20)

 6340136 reads
 5641782 (88.98%) aligned by nucmer -c 20 -l 20
 3453618 (54.47%) aligned by nucmer -c 32 -l 20
 2707005 (42.69%) aligned by nucmer -c 32 -l 32