Pseudodomonas syringae: Difference between revisions
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=== CBCB (new) === | === 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 | |||
=== Solexa assemblied for different read coverages === | === Solexa assemblied for different read coverages === | ||
Revision as of 21:08, 18 January 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
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
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
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