Pseudomonas aeruginosa: Difference between revisions
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I aligned the contigs to the reference. There seem to be no duplications in the new assembly !!! | I aligned the contigs to the reference. There seem to be no duplications in the new assembly !!! | ||
--- | |||
Other alignments: | |||
nucmer_params #alignments | |||
-l 17 -c 17 7261629 | |||
-l 20 -c 20 6541682 | |||
-l 14 -c 28 5178816 | |||
-l 28 -c 28 4950683 -l 14 -c 14 8307164 | |||
"-c 28" looks too stringent; many reads don't align | |||
"-l 14 -c 14" takes a very long time to run. If we decide to use these parameters, in order to speed up the process we could run nucmer twice: once using a larger match length (-l 16..20) and then a smaller match length (-l 14) on the reads which did not align or on the no/low coverage reference regions. The two delta files could be easily merged in the end and used as input for the layout. | |||
Revision as of 20:58, 15 January 2008
Strain PA-b1
Data
NCBI
Complete strains:
PAO1 AE004091 1 chromosome, 6,264,404 bp, 66.56 %GC PA14 CP000438 1 chromosome, 6,537,648 bp, 66.29 %GC PA7 CP000744 1 chromosome, 6,588,339 bp
CBCB
File location:
/fs/szasmg2/Bacteria/Pseudomonas_aeruginosa/
Files:
s_1_0001_prb.txt s_1_sequence.txt # contains seq & qual s_1_tag.txt s_7_0300_prb.txt s_7_sequence.txt # contains seq & qual s_7_tag.txt wc -l s_1_sequence.txt s_7_sequence.txt 4105993 s_1_sequence.txt 4521907 s_7_sequence.txt 8627900 total
grep -c N s_*seq s_1_sequence.seq:37933 s_7_sequence.seq:69669
Assemblies
Untrimmed reads: all contain ref duplications
1. 2007_1203_AMOSCmp-PAO1 2. 2007_1203_AMOSCmp-PAO1-relaxed 3. 2007_1204_AMOSCmp-PA14 4. 2007_1204_AMOSCmp-PA14-relaxed 5. 2007_1210_AMOSCmp_PA7-relaxed 6. 2007_1204_AMOSCmp-PA14-relaxed-noN (not shown): removed all reads that contain N's; same parameters as 2007_1204_AMOSCmp-PA14-relaxed => no big differences compared to 2007_1204_AMOSCmp-PA14-relaxed
QC stats:
Assembly 1 2 3 4 5 =================================================================================================== [Info] REF PAO1 PAO1 PA14 PA14 PA7 MINCLUSTER 20 20 20 20 20 MINOVL 10 3 10 3 3 MAJORITY 70 50 70 50 50 MINMATCH 20 20 20 20 20 [Scaffolds] TotalScaffolds 1 1 1 1 1 TotalContigsInScaffolds 4412 2197 3226 1828 25419 MeanContigsPerScaffold 4412 2197 3226 1828 25419 MinContigsPerScaffold 4412 2197 3226 1828 25419 MaxContigsPerScaffold 4412 2197 3226 1828 25419 TotalBasesInScaffolds 6000280 5985597 6137031 6127761 4924485 MeanBasesInScaffolds 6000280 5985597 6137031 6127761 4924485 MaxBasesInScaffolds 6000280 5985597 6137031 6127761 4924485 N50ScaffoldBases 6000280 5985597 6137031 6127761 4924485 TotalSpanOfScaffolds 6264430 6264430 6537674 6537674 6588355 MeanSpanOfScaffolds 6264430 6264430 6537674 6537674 6588355 MinScaffoldSpan 6264430 6264430 6537674 6537674 6588355 MaxScaffoldSpan 6264430 6264430 6537674 6537674 6588355 IntraScaffoldGaps 4411 2196 3225 1827 25418 2KbScaffolds 1 1 1 1 1 2KbScaffoldSpan 6264430 6264430 6537674 6537674 6588355 2KbScaffoldPercent 100 100 100 100 100 MeanSequenceGapSize 59 126 123 223 64 [Contigs] TotalContigs 4412 2197 3226 1828 25419 TotalBasesInContigs 6620035 6602214 6791916 6782045 5334464 MeanContigSize 1500 3005 2105 3710 210 MinContigSize 33 33 33 33 33 MaxContigSize 21576 46268 61425 86811 13454 N50ContigBases 3548 8478 6480 14280 410 [BigContigs_greater_10000] TotalBigContigs 44 173 142 229 3 BigContigLength 547699 2709766 2127747 4558923 34975 MeanBigContigSize 12448 15663 14984 19908 11658 MinBigContig 10018 10045 10022 10040 10262 MaxBigContig 21576 46268 61425 86811 13454 BigContigsPercentBases 8 41 31 67 0.66 [SmallContigs] TotalSmallContigs 4368 2024 3084 1599 25416 SmallContigLength 6072336 3892448 4664169 2223122 5299489 MeanSmallContigSize 1390 1923 1512 1390 209 MinSmallContig 33 33 33 33 33 MaxSmallContig 9964 9996 9977 9995 8411 SmallContigsPercentBases 92 59 69 33 99 [Reads] TotalReads 8627900 8627900 8627900 8627900 8627900 ReadsInContigs 6218126 6218255 6541160 6541364 3766798 BigContigReads 549658 2601986 2115038 4460608 39074 SmallContigReads 5668468 3616269 4426122 2080756 3727724 SingletonReads 2409774 2409645 2086740 2086536 4861102
1: nucmer -c 20 -l 20 2: nucmer -c 19 -l 19 3: nucmer -c 18 -l 18 4: nucmer -c 17 -l 17 5: nucmer -c 16 -l 16 : still running
Assembly 1 2 3 4 ======================================================================================== [Info] REF PA14 PA14 PA14 PA14 MINCLUSTER 20 19 18 17 MINMATCH 20 19 18 17 MINOVL 3 3 3 3 MAJORITY 50 50 50 50 [Scaffolds] TotalScaffolds 1 1 1 1 TotalContigsInScaffolds 1828 1760 1949 2447 MeanContigsPerScaffold 1828 1760 1949 2447 MinContigsPerScaffold 1828 1760 1949 2447 MaxContigsPerScaffold 1828 1760 1949 2447 TotalBasesInScaffolds 6127761 6139966 6161019 6202155 MeanBasesInScaffolds 6127761 6139966 6161019 6202155 MaxBasesInScaffolds 6127761 6139966 6161019 6202155 N50ScaffoldBases 6127761 6139966 6161019 6202155 TotalSpanOfScaffolds 6537674 6537675 6537676 6537680 MeanSpanOfScaffolds 6537674 6537675 6537676 6537680 MinScaffoldSpan 6537674 6537675 6537676 6537680 MaxScaffoldSpan 6537674 6537675 6537676 6537680 IntraScaffoldGaps 1827 1759 1948 2446 2KbScaffolds 1 1 1 1 2KbScaffoldSpan 6537674 6537675 6537676 6537680 2KbScaffoldPercent 100 100 100 100 MeanSequenceGapSize 223 225 192 136 [Contigs] TotalContigs 1828 1760 1949 2447 TotalBasesInContigs 6782045 7000867 7473670 8558383 MeanContigSize 3710 3978 3835 3498 MinContigSize 33 33 33 33 MaxContigSize 86811 99026 134098 254819 N50ContigBases 14280 20312 30580 52845 [BigContigs_greater_10000] TotalBigContigs 229 218 221 193 BigContigLength 4558923 5260728 6380541 7854838 MeanBigContigSize 19908 24132 28871 40699 MinBigContig 10040 10014 10072 10051 MaxBigContig 86811 99026 134098 254819 BigContigsPercentBases 67 75 85 92 [SmallContigs] TotalSmallContigs 1599 1542 1728 2254 SmallContigLength 2223122 1740139 1093129 703545 MeanSmallContigSize 1390 1128 633 312 MinSmallContig 33 33 33 33 MaxSmallContig 9995 9978 9849 9820 SmallContigsPercentBases 33 25 15 8 [Reads] TotalReads 8627900 8627900 8627900 8627900 ReadsInContigs 6541364 6744255 6967026 7260781 BigContigReads 4460608 5102815 5993784 6716141 SmallContigReads 2080756 1641440 973242 544640 SingletonReads 2086536 1883645 1660874 1367119
:::::::::::::: contig.summary :::::::::::::: -c,-l #elem #elem0 #elem<0 min median max sum mean stdev n50 20 1828 0 0 33 251 64109 5521501 3020.51 6134.05 11727 19 1760 0 0 33 129 94246 6659370 3783.73 9299.94 19361 18 1949 0 0 33 63 126592 7021203 3602.46 10698.9 29219 17 2447 0 0 33 52 234167 7908046 3231.73 13190.4 49318
:::::::::::::: contig.10000.summary :::::::::::::: -c,-l #elem #elem0 #elem<0 min median max sum mean stdev n50 20 178 0 0 10056 14356 64109 3173401 17828.1 9644.73 17994 19 204 0 0 10026 18436 94246 4868508 23865.24 15842.96 27908 18 212 0 0 10196 21088 126592 5899614 27828.37 19240.29 35458 17 190 0 0 10058 27883 234167 7223145 38016.55 30263.25 54897
2007_1204_AMOSCmp-PA14-relaxed
Find adjacent contigs:
1. nucmer contigs, filter adjacent contig end matches
overlaps haev to be >=20 bp => 12 overlaps
$ nucmer -c 20 -l 20 /fs/szasmg2/Bacteria/Pseudomonas_aeruginosa/Assembly/2007_1204_AMOSCmp-PA14-relaxed/nucmer/Pa.fasta /fs/szasmg2/Bacteria/Pseudomonas_aeruginosa/Assembly/2007_1204_AMOSCmp-PA14-relaxed/nucmer/Pa.fasta ...
[S1] [E1] | [S2] [E2] | [LEN 1] [LEN 2] | [% IDY] | [LEN R] [LEN Q] | [COV R] [COV Q] | [TAGS]
===============================================================================================================================
6738 6765 | 1 28 | 28 28 | 100.00 | 6765 41748 | 0.41 0.07 | 1132 1133 [END]
133 161 | 1 29 | 29 29 | 100.00 | 161 10235 | 18.01 0.28 | 1271 1272 [END]
11534 11631 | 1 98 | 98 98 | 92.86 | 11631 3390 | 0.84 2.89 | 1409 1410 [END]
102 122 | 1 21 | 21 21 | 100.00 | 122 46 | 17.21 45.65 | 1464 1465 [END]
281 301 | 1 21 | 21 21 | 100.00 | 301 183 | 6.98 11.48 | 1623 1624 [END]
659 988 | 1 303 | 330 303 | 79.15 | 988 418 | 33.40 72.49 | 1626 1627 [END]
177 416 | 5 241 | 240 237 | 75.93 | 418 1604 | 57.42 14.78 | 1627 1628 [END]
1296 1344 | 1 49 | 49 49 | 100.00 | 1344 15090 | 3.65 0.32 | 217 218 [END]
319 652 | 1 212 | 334 212 | 60.30 | 652 322 | 51.23 65.84 | 314 315 [END]
13265 13285 | 1 21 | 21 21 | 100.00 | 13285 14465 | 0.16 0.15 | 757 758 [END]
892 995 | 5 176 | 104 172 | 59.30 | 1000 3085 | 10.40 5.58 | 815 816 [END]
2406 2425 | 1 20 | 20 20 | 100.00 | 2425 5200 | 0.82 0.38 | 93 94 [END]
2. nucmer PA14 contigs to PAO1 contigs
Duplication causes
1. Only the reference nucmer alignment coordinates are stored in the bank and used by casm-layout and make-consensus; the read alignment coordinates are not stored, just the read clear ranges (the ones given in the .afg file); read positions in the assembly layout are approximate 2. reads can be shifted from their original alignment position by make-consensus by up to 2^3*15=120 bp (constants ALIGN_WIGGLE=15; MAX_ALIGN_ATTEMPTS=3) 3. The Pseudomonas_aeruginosa reference contains multiple 2 copy 5-10 bp kmers, adjacent within a few dozen bp of each other; reads starting with these kmers align in 2 ways to the reference; if the reads contain errors or SNP's, the 2nd (shorter) alignment to the greedyly built consensus is chosen over the correct one and a duplication is introduced
Trimmed reads
Quality trimming
Art's script: qual-trim.awk
Min_Qual=15
. #elem #elem0 #elem<0 min median max sum mean stdev n50 qualTrim 8627900 1266 0 0 23 33 193999902 22.49 7.43 26
!!! Avg clr drops from 33 to 22 bp !!! Duplications still exist though at a lower rate
Alignment based trimming
!!! Reduced the dupplications significantly
Solution:
1. align all reads to the reference using nucmer. I initially used minmatch=17, mincluster=17 (-c 17 -l 17)
7.26M total reads align to the reference
5.32M reads aligned on their full length (as opposed to 0.94M
33bp quality untrimmed reads)
2. trim reads according to their nucmer alignment coordinates 3. assemble them using the AMOScmp pipeline. A modified version of make-consensus was used (ALIGN_WIGGLE=2 instead of 15)
2008_0109_AMOSCmp-PA14-relaxed-17-nucmer
command: /nfshomes/dpuiu/bin/AMOScmp Pa -D MINCLUSTER=17 -D MINMATCH=17 -D MINOVL=5 -D MAJORITY=50 -D ALIGNWIGGLE=2 location: /fs/szasmg2/Bacteria/Pseudomonas_aeruginosa/Assembly/2008_0109_AMOSCmp-PA14-relaxed-17-nucmer qc stats: /fs/szasmg2/Bacteria/Pseudomonas_aeruginosa/Assembly/2008_0109_AMOSCmp-PA14-relaxed-17-nucmer/Pa.qc
#contigs 3137 #singleton_contigs 1106 (probably kmers that match by chance?) min 17 1quater 17 median 27 3quater 114 max 103575 sum 6168056 mean 1966.23 stdev 7084.82 n50 22308
I aligned the contigs to the reference. There seem to be no duplications in the new assembly !!!
---
Other alignments:
nucmer_params #alignments -l 17 -c 17 7261629 -l 20 -c 20 6541682 -l 14 -c 28 5178816 -l 28 -c 28 4950683 -l 14 -c 14 8307164
"-c 28" looks too stringent; many reads don't align "-l 14 -c 14" takes a very long time to run. If we decide to use these parameters, in order to speed up the process we could run nucmer twice: once using a larger match length (-l 16..20) and then a smaller match length (-l 14) on the reads which did not align or on the no/low coverage reference regions. The two delta files could be easily merged in the end and used as input for the layout.