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Completing microbial genome assemblies: strategy and performance comparisons
Determining the genomic sequences of microorganisms is the basis and prerequisite for understanding their biology and functional characterization. While the advent of low-cost, extremely high-throughput second-generation sequencing technologies and the parallel development of assembly algorithms have generated rapid and cost-effective genome assemblies, the assemblies are often unfinished, fragmented draft genomes as a result of the short read lengths and long repeats present in multiple copies. Several methods, such as ALLPATH-LG, hybrid and non-hybrid approaches, have been proposed to utilize the third-generation sequencing long reads that can span many thousands of bases for complete microbial genome assemblies. However, there appears an insufficiency on standardized procedure for strategy comparison and evaluation on their assemblies.
In this article, we provide a comprehensive review of the above-motioned methods, and collect datasets for comparative assessment of the non-hybrid approaches—hierarchical genome-assembly process (HGAP) and self-correction approach (SCA). In addition to offering explicit and useful recommendations to practitioners, the review guides to design a project in finishing microbial genome assembly. Following a special recipe proposed by ALLPATHS-LG, to supply it with the three prepared libraries—fragment, jump and long reads, ALLPATHS-LG is able to complete microbial genomes as the sequencing coverage is controlled at 100X. Although the hybrid approach could improve the continuity over the assembly produced by the next-generation sequencing reads along, we did not successfully assemble a complete genome. The both non-hybrid approaches—HGAP and SCA—are able to produce complete genomes as long as the third generation sequencing reads are adequately long and sufficient.
Datasets employed in this study
We have used ALLPATHS-LG to assemble three bacterial genomes: E. coli, R. sphaeroides, and S. pneumoniae. The sequencing reads for these three genome assemblies are summarized in the following table (D1-D3).
We have conducted a hybrid approach proposed by Koren et al.(ref) to correct long reads (D5) with short reads (D4) (by PacBioToCA), then to de novo assemble the corrected long reads (by runCA) for E. coli genome reconstruction.
We have conducted the both non-hybrid approaches: hierarchical genome-assembly process (HGAP) and self-correction approach (SCA) to de novo assemble the PacBio long reads. The datasets used in the non-hybrid approach are composed of various SMRT cells, ranging from 4 to 17 XL-C2 SMRT cells (D5-D8), and a single SMRT cell gathered with PacBio RS II system and P4-C2 chemistry (D9).
| Data | Organism | Fragment | Jump | Long read | Reference |
| D1 | E. coli K-12 MG1655 | 2×101 bp, 180 bp insert (SRR447685) | 2×93 bp, 3000 bp insert (SRR401827 and SRR492488) | 1-3 Kbp (Ribeiro's ftpa) | NC_000913 |
| D2 | R. sphaeroides 2.4.1 | 2×101 bp, 180 bp insert (SRR125492) | 2×101 bp, 3000 bp insert (SRR388672) | 1-3Kbp (Ribeiro's ftpa) | NC_007488-90, NC_007493-94, NC_009007-08 |
| D3 | S. pneumoniae Tigr4 | 2×101 bp, 180 bp insert (SRR387335) | 2×93 bp, 3000 bp insert (SRR364158) | 1-3 Kbp (Ribeiro's ftpa) | NC_003028 |
| D4 | E. coli K-12 MG1655 | 2×151 bp, 300 bp insert (Illumina data websiteb) | NC_000913 | ||
| D5 | E. coli K-12 MG1655 | 10 Kbp, 17 SMRT cell (SRX255228c) | NC_000913 | ||
| D6 | E. coli K-12 MG1655 | 8-10 Kbp, 8 SMRT cells (SRX260475d) | NC_000913 | ||
| D7 | M. ruber DSM1279 | 8-10 Kbp, 4 SMRT cells (SRX260496d) | NC_013946 | ||
| D8 | P. heparinus DSM2366 | 8-10 Kbp, 7 SMRT cells (SRX260506d) | NC_013061 | ||
| D9 | E. coli K-12 | PacBio RS II System and P4-C2 chemistrye, 20 Kbp library, 1 SMRT cell | NC_000913 |
a Long reads were downloaded from ftp://ftp.broadinstitute.org/pub/papers/assembly/Ribeiro2012/data
b Paired reads were provided in http://www.illumina.com/systems/miseq/scientific_data.ilmn
c PacBio HDF5 files were requested from NCBI Sequence Read Archive (SRA).
d PacBio HDF5 files were downloaded from http://files.pacb.com/software/hgap/index.html
e PacBio HDF5 files were downloaded from https://github.com/PacificBiosciences/DevNet/wiki/E.-coli-20kb-Size-Selected-Library-with-P4-C2