E. coli

Revision as of 19 April 2012 00:36 by admin (Comments | Contribs) | (→Contig integrator)

Escherichia coli K12 MG1655. The E. coli MG1655 consists of a circular chromosome of 4,639,675 bp in length.

Read source

The illuminia read data of E. coli (Paired-end sequencing library with 200 bp inserts) were downloaded from Sequence Read Archive (SRA). More than 20.8 M reads

Sequence assembly

  • Set1 (Different Assemblers)
Software Version Parameters Download
ABySS 1.3.0 k=31 Abyss
Velvet 1.1.04 k=29 ins_length=215 cov_cutoff=12 exp_cov=24 min_contig_lgth=100 scaffolding=no Velvet
Edena 3 m=30 Edena
SOAPdenovo 1.05 K=29 M=3 SOAPdenovo
CLC 4.7.2 insert_size_range=194,236 minimum_contig_length=100 CLC

Merged File: Set1_Contig

The above assemblers together with the parameter setting have been selected for de novo assembling of E. coli. After assembly, we discarded contigs with less than 100bp and evaluated the accuracy of the assemblies based on the Mauve Assembly Matrices (the results are shown below). In this set of data, we have different sequence assemblies, each was generated by the different assembler. Because the same assembler performs differently over varying parameter settings, such as kmer, we have tried different parameters for Abyss and SOAPdenovo in the following sets.
  • Set2 (Different parameters for Abyss - the assembler provides the lowest number of contigs in Set1)
Abyss parameter Download
k=29 Abyss_k29
k=31 Abyss_k31
k=33 Abyss_k33

Merged File: Set2_Contig

  • Set3 (Different parameters for SOAPdenovo - the assembler provides the largest number of contigs in Set1)
SOAPdenovo parameter Download
k=29 SOAP_k29
k=31 SOAP_k31
k=33 SOAP_k33

Merged File: Set3_Contig


  • Scaffold the contigs using SSPACE
Download
CISA+SSPACE
ABySS+SSPACE
Minimus2+SSPACE

Contig integrator

  • CISA
Input Download
Set1 CISA_Set1
Set2 CISA_Set2
Set3 CISA_Set3
Set2+Set3 CISA_Set2_3
Set1+2+3+2_3 CISA_Set1+2+3+2_3

The input file for CISA is an assembled set of contigs, e.g., the set1 contains the contigs obtained from Abyss, CLC, Edena, SOAPdenovo, and Velvet.

The integrated contigs generated by CISA can be directly downloaded via the Download link.

  • minimus2
Input Download
Set1 minimus2_Set1(A,C,E,S,V)
Set1 minimus2_Set1(A,C,E,S,V)

Evaluation

  • Benchmark genome
Eshcherichia coli K12 MG1655
  • Evaluate by Mauve Assembly Metrics
How to score genome assemblies using the Mauve system
  • Score with Mauve metrics:

Set1

Name NumContigs NumAssemblyBases NumMisCalled NumUnCalled NumGapsRef NumGapsAssembly TotalBasesMissed PercBasesMissed ExtraBases PercExtraBases BrokenCDS IntactCDS ContigN50 ContigN90 MaxContigLength Blast_IntactCDS
Abyss 133 4626205 334 69 123 119 57847 1.2468 29424 0.636 57 4263 96157 26096 222425 4257
CLC 379 4546926 100 0 288 287 130550 2.8138 3405 0.0749 62 4258 29767 8447 107342 4233
Edena 211 4569446 17 0 129 125 86780 1.8704 2078 0.0455 66 4254 54405 13642 186686 4204
SOAPdenovo 553 4547211 36 0 461 412 124407 2.6814 6972 0.1533 100 4220 17902 5384 103369 4146
Velvet 283 4550675 138 0 208 203 116542 2.5119 2783 0.0612 74 4246 52474 12537 166094 4204
CISA_Set1 72 4626972 243 50 91 90 49312 1.0628 31326 0.677 45 4275 119107 32288 310578 4290
minimus2 74 4608653 285 0 97 78 76881 1.657 35464 0.7695 50 4270 126075 34542 417704 4268
We have visually inspected the assemblies against the reference genome (NC_000913) by using graphic representations, e.g. dot plots. Therefore, we knew that the largest contig generated by minimus2 was misassembled.
Dotplot.jpg

Set2-Set3

Name NumContigs NumAssemblyBases NumMisCalled NumUnCalled NumGapsRef NumGapsAssembly TotalBasesMissed PercBasesMissed ExtraBases PercExtraBases BrokenCDS IntactCDS ContigN50 ContigN90 MaxContigLength Blast_IntactCDS
Abyss_k29 130 4634010 322 30 118 115 61835 1.3327 40405 0.8719 54 4266 95691 26567 268182 4267
Abyss_k31 133 4626205 334 69 123 119 57847 1.2468 29424 0.636 57 4263 96157 26096 222425 4257
Abyss_k33 135 4644184 354 338 139 119 66355 1.4302 44937 0.9676 78 4242 89001 24907 268398 4263
CISA_Set2 106 4635666 327 146 116 102 55420 1.1945 39743 0.8573 64 4256 113377 27272 222663 4269
SOAP_k29 1373 4582756 48 0 466 415 124372 2.6806 7247 0.1581 100 4220 17892 5276 103369 4146
SOAP_k31 1295 4583165 56 0 510 466 121606 2.621 9201 0.2008 121 4199 17003 4286 77302 4094
SOAP_k33 2170 4608265 105 0 1470 1380 126273 2.7216 41165 0.8933 507 3813 5391 1449 22953 3379
CISA_Set3 440 4532901 41 0 379 338 132794 2.8621 5627 0.1241 87 4233 23332 6355 103369 4166
CISA_Set2&3 105 4636950 350 160 118 104 55228 1.1903 40097 0.8647 61 4259 113377 27272 222663 4269
CISA_Set_1_2_3_2&3 72 4637760 521 53 109 97 43006 0.9269 37427 0.807 44 4276 115185 35678 310691 4291
As can be seen here, CISA can successfully integrate the sets of contigs and reduce the number of contigs. However, in comparison with integrating assemblies generated from different assemblers (Set 1), varying assemble parameters is less efficient (in the case of Set 2 or Set3) in completing the genome.
  • Scaffold the contigs using SSPACE
Since we have the paired-end reads of E. coli, it is possible to assess the order, distance and orientation of contigs and combine them into scaffolds. We, therefore, used SSPACE to scaffold the contigs and quantified the scaffolds by Mauve assembly metrics.
Name NumContigs NumAssemblyBases NumMisCalled NumUnCalled NumGapsRef NumGapsAssembly TotalBasesMissed PercBasesMissed ExtraBases PercExtraBases BrokenCDS IntactCDS ContigN50 MaxContigLength Blast_IntactCDS
CISA+SSPACE 69 4627290 237 50 94 93 52804 1.1381 37397 0.8082 44 4276 134584 416708 4290
Abyss+SSPACE 101 4627104 393 735 114 119 54956 1.1845 33747 0.7293 57 4263 107040 268750 4272
minimus2+SSPACE 64 4608774 337 54 93 76 75502 1.6273 36021 0.7816 49 4271 150458 420117 4268
The results show:
  1. The integrated contigs output by CISA can be scaffolded by SSPACE to a limited extent (from 72 to 69), which suggests that our CISA can indeed integrate the sequence information from different assemblies.
  2. To introduce the paired-end reads to the pre-assembled contigs generated by Abyss using SSPACE (Abyss+SSPACE) can only reduce the number of contigs from 133 to 101, smaller than the effect made by CISA (from 133 to 72), which suggests that contig integration prior to scaffolding can further enhance the result.
  3. The problem of misassembled contigs generated by minimus2 is not yet solved by SSPACE, which suggests that we should integrate contigs with caution.