LOCAS - A Low Coverage Assembler for Resequencing Figure 4. Performance comparison of homology-guided assembly on simulated data. We simulated a resequencing study of an artificial A. thaliana strain using a sequencing depth of 7.56. The simulated Illumina reads were aligned to the reference genome Col-0 and partitioned into blocks of 25 kb using SHORE. The assembly tools SUPERLOCAS and VELVET were applied to assemble the mapped reads of the first chromosome and the left-over reads. The avgN50 (average N50) is plotted against the avgERR (average error rate) for the assembly tools SUPERLOCAS and VELVET (in left-over incorporation mode). SUPERLOCAS is displayed in red and VELVET in blue. doi:10.1371/journal.pone.0023455.g004 reads aligning one of the blocks separately by incorporating high quality left-over reads in the assembly. Thus, the tool can assemble very long insertions of several kb located between two highly homologous regions. In order to reduce the complexity of the overlap graph, different alignment constraints can be set as a function of the distance between reads in the reference genome. Performance Evaluation We evaluated the performance of LOCAS in three studies. The first study simulated de novo assemblies of small genomic regions and compared the performance of current NGS assemblers for data with a low sequencing depth. For the second study, we simulated a homology-guided assembly of a divergent strain of Arabidopsis thaliana, again at low sequencing depth. For the third study, we performed a homology-guided assembly using Illumina reads from the Arabidopsis thaliana 1001 Genomes Project (http:// 1001genomes.org/). For the first study, we compared LOCAS with the publicly available short read assembly tools VELVET [14], EULER-SR [15], ABySS [16,21] and SOAPdenovo [22]. In the second and third studies, we compared SUPERLOCAS (LOCAS extension for homology-guided assembly) only with VELVET, since the other assemblers (ABySS, SOAPdenovo and EULER-SR) did not perform well enough considering contig length and error rate in the first study. Even though EDENA [23] also uses an overlap-layout-consensus approach, we did not consider it in our evaluation, because it does not utilize paired read information, does not allow for mismatches in the overlap calculation and produced insufficient results for low sequencing depth assemblies. Homology-guided assemblers like AMOScmp-shortReads (un- published) and its predecessor AMOScmp [24] provide an integrated solution using alignments to the reference in both the overlap as well as the consensus and scaffolding step, while LOCAS relies on pre-computed alignments from third party tools and does not use homology information in the consensus or scaffolding step of the assembly. Thus resulting contigs are hardly comparable without further post-processing as for instance implemented in the SHORE homology-guided assembly frame- work [25] combining LOCAS and other short read assemblers with the scaffolding tool BAMBUS [26] and quality assessment based on re-alignments of original reads to novel contigs. All datasets used for evaluation are available at http://www-ab. informatik.uni-tuebingen.de/software/locas. Evaluation of Low Sequencing Depth Assembly We used the first chromosome of A. thaliana Col-0 for our performance studies. First, we simulated Illumina GAIIx reads at a sequencing depth of 7.56 using METASIM [21]. We chose this particular sequencing depth since it is the lowest in which the assemblers showed reasonable contig sizes. The reads were assigned to reference sequence corresponding to their origin positions and partitioned into blocks of 10 kb length. Assemblies of PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23455