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, such 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 to facilitate the assembly of complete microbial genomes. However, standardized procedures that aim at evaluating and comparing these approaches are currently insufficient in the context of assembly large libraries of genomic data.

In this article, we provide a comprehensive review of the above-motioned methods and collect datasets for the 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 aims to aid in the design of a paradigm positioned to complete microbial genome assembly. Following a special methodology proposed by ALLPATHS-LG, the algorithm is supplied with three pre-prepared libraries—fragment, jump and long reads. ALLPATHS-LG is subsequently able to complete microbial genomes as the sequencing coverage is controlled at 100X. Although the hybrid approach could improve continuity over the assembly produced by second-generation sequencing reads, we remained unsuccessful in the completion of a complete genome using this approach. Both non-hybrid approaches—HGAP and SCA—are able to produce complete genomes provided that the third generation sequencing reads are adequately long and complete.

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 firstly investigated the effect of sequencing depths on assembly (Read Depths), then set genome size in running pacBioToCA, finally we tried different Celera Assembler parameters for runCA.

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).

^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