Culex pipiens symbiont
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Data Sources
Sanger: Wolbachia pipientis endosymbiont of Culex quinquefasciatus
Old reference: file name: /fs/szasmg2/Culex_pipiens_symbiont/Sanger/Wb_Cq_061226.dbs
Top 10 seqs Name Length %GC culex173d08.p1k 1457497 34.17 culexbac1d10Bg07.p1k 24726 35.11 culex3d09.p1k 15587 21.81 culex166f03.q1k 13962 36.17 culex_1177_1189-1a02.w2k1177 13564 37.10 culex26b07.p1k 9245 35.53 culex174d04.p1k 8832 33.64 J28015Ag08.q1ka 7809 36.04 culex180e07.p1k 6960 36.59 culex53a02.p1k 5343 33.58 ...
New reference (12 sequences): file name: /fs/szasmg2/Culex_pipiens_symbiont/Sanger/Wb_Cq.dbs
All seqs:
Name Length %GC
1 culexbac1b5Ab03.q1k 1136301 34.17
2 culex161b01.q1k 346054 34.25
3 culex166f03.q1k 13962 36.17 share almost all sequence with culex161b01.q1k & 1996bp with culexbac1b5Ab03.q1k
subtotal(3) 1496317
4 culex49c07.p1k 9245 35.53 looks circular(misoriented mates at the ends); region 4979-6364 aligns to culexbac1b5Ab03.q1k 3 times
5 culex53a02.p1k 5343 33.58 ~ 1Kbp alignments to culexbac1b5Ab03.q1k & culex161b01.q1k
6 culex117e02.p2kA55 3501 33.10 contained (in 2 pieces) in culexbac1b5Ab03.q1k
7 culex141a08.q1k 1920 33.44 few hundred bp alignments to other culex* seqs
subtotal(7) 1516326
8 culex180e07.p1k 6960 36.62 "CONTAINED" culexbac1b5Ab03.q1k (surrogate in WGA)
9 culex5c05.p1k 15587 21.81 low GC%; no alignments to NC_002978 & NC_006833; best hit is Anopheles gambiae complete mitochondrial genome : 15363 bp (96% coverage, 86% max id)
10 culex14h11.p1k 3350 51.73 repeat (higher GC%): good cvg of culex 18SrRNA gene ; no alignments to NC_002978 & NC_006833
11 culex22h10.q1k 2148 54.89 repeat (higher GC%): some alignment to culex 118S rRNA ; no alignments to NC_002978 & NC_006833
12 culex166d08.p1k 2071 55.53 repeat (higher GC%): culex 18S rRNA & 28S rRNA ; no alignments to NC_002978 & NC_006833
total(12) 1546442
NCBI:
- Genome Project
- TA : 7,379,314 traces (Sept 2007)
SEQ_LIB_ID SIZE STDEV CENTER_NAME TYPE COUNT PERCENT 1099499586718 9000 2700 TIGR_JCVIJTC WGS 15349 0.21 1099522705601 3500 1050 TIGR_JCVIJTC WGS 16116 0.22 1099641499000 33000 9900 TIGR_JCVIJTC WGS 768 0.01 G766BES1 120000 . WIBR WGS 100434 1.37 BE G771K1 5000 500 WIBR CLONEEND 51540 0.7 G772K1 5000 500 WIBR CLONEEND 25314 0.34 G809K1 2000 200 WIBR CLONEEND 29949 0.41 G810K1 2000 200 WIBR CLONEEND 2295 0.03 G818F1 40000 4000 WIBR WGS 437994 5.96 G818F2 40000 4000 WIBR WGS 8505 0.12 G818P1 4000 400 WIBR WGS 580557 7.89 G818P2 4000 400 WIBR WGS 1091326 14.84 G818P3 4000 400 WIBR WGS 350523 4.77 G818P4 4000 400 WIBR WGS 1017105 13.83 L31420P2 5000 . WIBR SHOTGUN 2259 0.03 L31422P1 4000 . WIBR SHOTGUN 3766 0.05 L31424P2 5000 . WIBR SHOTGUN 2226 0.03 L31425P1 4000 . WIBR SHOTGUN 3817 0.05 L31426P1 4000 . WIBR SHOTGUN 2274 0.03 L31427P1 4000 . WIBR SHOTGUN 2273 0.03 L31428P1 4000 . WIBR SHOTGUN 3045 0.04 L31429P1 4000 . WIBR SHOTGUN 3034 0.04 L31430P1 4000 . WIBR SHOTGUN 2261 0.03 L31431P1 4000 . WIBR SHOTGUN 2918 0.04 L31432P1 4000 . WIBR SHOTGUN 2947 0.04 L31433P1 4000 . WIBR SHOTGUN 2251 0.03 L31435P1 4000 . WIBR SHOTGUN 2987 0.04 L31439P1 4000 . WIBR SHOTGUN 2292 0.03 L31440P1 4000 400 WIBR SHOTGUN 1478 0.02 L31440P1 4000 . WIBR SHOTGUN 2281 0.03 L31441P1 4000 . WIBR SHOTGUN 2297 0.03 L31444P2 5000 . WIBR SHOTGUN 2241 0.03 L31446P2 5000 . WIBR SHOTGUN 2278 0.03 L31448P1 4000 . WIBR SHOTGUN 3052 0.04 L31449P1 4000 . WIBR SHOTGUN 2234 0.03 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_01-G-CULEX-10KB 10000 2000 TIGR_JCVIJTC WGS 1939130 26.36 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_02-G-CULEX-4KB 4000 800 TCAG_JCVIJTC WGS 119990 1.63 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_02-G-CULEX-4KB 4000 800 TIGR_JCVIJTC WGS 213407 2.9 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_03-F-CULEX-40KB 40000 8000 TCAG_JCVIJTC WGS 2405 0.03 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_03-F-CULEX-40KB 40000 8000 TIGR_JCVIJTC WGS 101370 1.38 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_04-F-CULEX-40KB 40000 8000 TCAG_JCVIJTC WGS 16126 0.22 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_04-F-CULEX-40KB 40000 8000 TIGR_JCVIJTC WGS 22134 0.3 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_05-F-CULEX-40KB 40000 8000 TIGR_JCVIJTC WGS 51281 0.7 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_06-G-CULEX-10KB 11000 2200 TIGR_JCVIJTC WGS 992283 13.49 MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_07-G-CULEX-10KB 9000 1800 TIGR_JCVIJTC WGS 106326 1.45 . . . WIBR OTHER 229 0 . . . WIBR PCR 228 0 . . . WIBR TRANSPOSON 8096 0.11 Total 7354992 100
CENTER_NAME TRACE_TYPE_CODE COUNT PERCENT WIBR WGS 3586444 48.76 TIGR_JCVIJTC WGS 3458164 47.02 TCAG_JCVIJTC WGS 138521 1.88 WIBR CLONEEND 109098 1.48 WIBR SHOTGUN 54211 0.74 WIBR TRANSPOSON 8096 0.11 WIBR OTHER 229 0 WIBR PCR 228 0 Total 7354992 100
JCVI:
Articles
Other Strains (complete)
RefSeq GenBank Pub Length (Mbp) GC Prot RNAs Wolbachia endosymbiont of Drosophila melanogaster(TIGR) NC_002978 AE017196 1 1.26778 35.2% 1195 39 Wolbachia endosymbiont strain TRS of Brugia malayi srain wMel(NEB) NC_006833 AE017321 1 1.08008 34.2% 805 37 Wolbachia pipientis wPip(Sanger) NC_010981 AM999887 1 1.48246 34.2% 1275 37
!!! Wolbachia pipientis wPip(Sanger) = culex161b01.q1k(346,054) + N(102) + culexbac1b5Ab03.q1k(1,136,301)
Read Counts
query_tracedb "query count SPECIES_CODE='CULEX PIPIENS QUINQUEFASCIATUS'" # 7552113 : all traces query_tracedb "query count SPECIES_CODE='CULEX PIPIENS QUINQUEFASCIATUS' AND load_date >='09/01/2007'" # 172799 : new traces (all cDNA)
Assembly
Locations:
/fs/szasmg2/Culex_pipiens_symbiont/
2006_1226_WGA
initial assembly
Steps:
1. All cpqg reads have been downloaded from the TA (July 2006). The reads have been grouped by libraries and the clear range has been computed. There were 6.6M reads in the download compared with 7.3M now. Unfortunately I've only noticed this difference at the end of my experiment.
2. The Wolbachia endosymbiont of Culex quinquefasciatus assembly has been downloaded from the Sanger ftp site ( ftp://ftp.sanger.ac.uk/pub/pathogens/Wolbachia/Wb_Cq.dbs ) ; there are 95 sequences in this file. Most of them are very short. Below are listed the name,length & gc% of the longest 10: name length(bp) gc% culex173d08.p1k 1457497 34.17 culexbac1d10Bg07.p1k 24726 35.11 culex3d09.p1k 15587 21.81 culex166f03.q1k 13962 36.17 culex_1177_1189-1a02.w2k1177 13564 37.10 culex26b07.p1k 9245 35.53 culex174d04.p1k 8832 33.64 J28015Ag08.q1ka 7809 36.04 culex180e07.p1k 6960 36.59 culex53a02.p1k 5343 33.58
3. The cpqg random reads (clr only) have been aligned to symbiont sequences using nucmer (default parameters)
4. The nucmer output has been analyzed. It's been noticed that many of the short symbiont sequences (2-3KB in length) have a higher than expected number of alignments. To avoid the repeats I've selected only the reads that aligned to the longest 10 symbiont sequences (see above).
5. A 95% identity and minimum of 400 bp alignment thold has been used to
determine the symbiont reads. There were 29,110 unique reads (30,690
reads+mates) selected. Below is a per library breakdown (reads+mates):
MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_01-G-CULEX-10KB 9581
MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_06-G-CULEX-10KB 4549
G818P4 3784
G818P2 3478
G818P1 2238
G818F1 1283
MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_02-G-CULEX-4KB 1156
MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_03-F-CULEX-40KB 738
G818P3 723
MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_07-G-CULEX-10KB 556
MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_05-F-CULEX-40KB 327
MSC-CULEX-PIPIENS-QUINQUEFASCIATUS_04-F-CULEX-40KB 185
1099522705601 99
G809K1 89 : cDNA , should be removed
1099499586718 77
G772K1 12 : cDNA , should be removed
G771K1 10 : cDNA , should be removed
G766BES1 4 : BE library
1099641499000 2
6. The reads have been assembled using the runCA-OBT.pl script (default parameters). Most of the reads got assembled into 3 large scaffolds. There is mate pair evidence (outie mates) that the largest scaffold is circular.
All the scaffolds ens up in surrogates (20-50KB total surrogate length) Are there not enough BE to span the unique regions?
Cpqg.qc
scaff_8 Longest scaff scaff_9 2nd longest scaff scaff_7 3rd longest scaff scaff_6 Small scaff that Looks circular
7. The scaffolds/contigs have been aligned to longest 10 Wolbachia endosymbiont sequences. Most of the long alignments were at over 99% identity. However, several large rearrangements have been noticed.
Wb_Cq-vs-scaff Reference vs scaff
2007_0802_WGA-default
new assembly
Steps:
1. All Culex reads have been downloaded from TA . ~1M new reads sincd 2006_1226
2. The reads have been aligned to the new reference (exclude mito,repeats) using nucmer (default parameters)
3. A 95% identity and minimum of 400 bp alignment thold has been used to determine the symbiont reads. 3850 new reads & mates in addition to the previous ones were identified
4. 33,783 reads have been assembled using the runCA-OBT.pl script (default parameters). Cpqg.qc
Compared to the initial assembly, many metrics went down (TotalBasesInScaffolds,MaxBasesInScaffolds,MaxContigLength ...) TotalSurrogates & SurrogateInstances more than doubled
2007_0802_WGA-0.5E
error rate =0.5 % => more fragmented assembly
2007_0802_WGA-0.5M
genome size=1.5M => more TotalBasesInScaffolds but more unhappy mates
What to do next?
- use CA 5.1 (latest version)
- remove 958 cDNA's aligned to culex*
- increase utg error rate to from 1.5% to 2% (3% gave worse results than 2%)
- recruite reads that align to contig ends: some ends are repetitive => too many; others no alignments
- use 2 other complete strains; only 2 new aligned reads were identified
- AMOScmp new reference => more unhappy mates then before
- dropping the min Astat from 1 to -1 made some degens into places ctgs; did not improve overall stats
- separate JCVI & WIBR reads, assemble separately => 5 obvious alignment breaks
- use only the reads from lib with insert size <=11Kbp => more fragmented
- use only the reads that aligned to the top2 Sanger ctgs (36606 instead of 36767)
Reads aligned to the 7 Sanger sequences:
CENTER STRATEGY COUNT PERCENTAGE TIGR_JCVIJTC WGS 18268 48.97 WIBR WGS 17316 46.42 # 155 BE align but mostly at 80-90% id, only 4 at >=95% id, >=400bp WIBR CLONEEND(CDNA) 884 2.37 # about avg 1.48 TCAG_JCVIJTC WGS 815 2.18 WIBR SHOTGUN 20 0.05 total 37303 100 total+mates 39027 (37724 in .frg file) wgs+mates 38069 (36767 in .frg file) # 302 BE
2008_0829_WGA-wgs
e=1.5
[Top5Scaffolds=contigs,size,span,avgContig,avgGap] 0=3,1363559,1364974,454520,708 1=1,53307,53307,53307,0 2=1,28821,28821,28821,0 3=2,23208,23528,11604,320 4=1,8315,8315,8315,0 total=8,1477210,1478945,184651,578
2008_0829_WGA-wgs e=2.0
16 scaffolds, 21 ctgs & 92 degens
[Top5Scaffolds=contigs,size,span,avgContig,avgGap] 0=4,1388064,1389477,347016,471 1=3,70356,70440,23452,42 2=1,42565,42565,42565,0 3=1,8315,8315,8315,0 4=1,2425,2425,2425,0 total=10,1511725,1513222,151172,299
Media:NC_010981-scf.filter-q.png Media:NC_010981-ctg-deg.filter-q.png Media:Cpqg.all.infoseq
100+ bp 0cvg regions in the reference:
1. culexbac1b5Ab03.q1k 429746 429972 226 0 2. culexbac1b5Ab03.q1k 907129 908079 950 0 1. NC_010981.1 775928 776047 120 0 2. NC_010981.1 1253284 1254139 856 0
1.1. NC_010981.1 RefSeq gene 775763 777826 . + . contains GeneID:6385213 # WP0709 Putative outer membrane protein
1.2. NC_010981.1 RefSeq gene 1252115 1253287 . + . begin GeneID:6385392 # tuf translation elongation factor tu
1.3. NC_010981.1 RefSeq gene 1253302 1253622 . + . contained GeneID:6385310 # rpsJ 30s ribosomal protein s10 ??? missing;
# very conserved in Wolbachia endosymbiont of Drosophila melanogaster
2.1. NC_010981.1 RefSeq gene 1253632 1254354 . + . end GeneID:6385679 # rplC ribosomal protein L3
# very conserved in several species : Wolbachia, Erlichia ...
No promer alignments of sequences to these regions
100+ bp 0cvg regions in the assembly:
ctg_start_stop len gc% comments 1. ctg7180000001230_202_1361 1160 37.50 # first 600 have no alignments; last 400bp are cloning vector 2. ctg7180000001305_11303_11867 565 33.45 # aligns at 100%len, 100%id to Wolbachia endosymbiont of Drosophila melanogaster, complete genome; NC_002978.6:243504..243803 3. ctg7180000001305_13367_14006 640 33.91 # good blastx alignment to Wolabchia gene on 100% length; NC_002978.6:488974..489912 4. deg7180000001252_328_851 524 35.88 # blastx align to We of Bm NC_006833.1:754520..755170
Identify more reads that align to those regions (blastn TA):
236 : all 198 : new 395 : new+mates
Adding these reads did not improve the assembly.
gi|42519920|ref|NC_002978.6| 243504 243803 # ctg7180000001305_11303_11867 565 33.45 : there are reads aligned to 241173-243822 gi|42519920|ref|NC_002978.6| 488974 489912 # ctg7180000001305_13367_14006 640 33.91 : there are reads aligned to 487182-492517 gi|58584261|ref|NC_006833.1| 754520 755170 # deg7180000001252_328_851 524 35.88 : there are reads aligned to 754300-755148
All the reads aligned to the 3 regions above have been assembled; the 3 regions seem to contain rearrangements