Culex pipiens symbiont

Data Sources

NCBI:

• Genome Project
• TA : 7,379,314 traces (Sept 2007)

Sanger: Wolbachia pipientis endosymbiont of Culex quinquefasciatus

• Genome Project
• FTP

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
 5  culex53a02.p1k          5343    33.58
 6  culex117e02.p2kA55      3501    33.10
 7  culex141a08.q1k         1920    33.44 
    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

JCVI:

• MSC

Articles

• Salzberg_GB_2005.pdf
• Sanger_2008

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?