speculate that the assembly produced by our approach might be less affected by repetitive regions than de novo assembly because some repeats are already solved during the alignment step. Since SUPERLOCAS explicitly treats left-over reads and aligned reads differently, and can reuse the left-over graph in multiple local assembly executions, the incorporation of left-over reads is more reliable and much faster than for other short read assemblers. We assume that in future, our method will gain from improvements in sequencing technologies. Our overlap-layout- consensus approach can benefit from longer reads as it is more robust to sequencing errors at the end of reads. Computing overlap alignments rather than using exact matches of sub-regions (like de Bruijn graph methods do) in order to overlap the reads will become more important as longer sub-matches have a higher probability of containing sequencing errors. In addition, the recruitment of left-over reads will become more reliable due to an increased overlap length between reads. Finally, the potential of homology-guided assemblies grows steadily with increasing numbers of completely sequenced genomes. Design and Implementation The software is implemented in C++ and supported on Linux. Availability Source code and binaries are freely available from our website http://www-ab.informatik.uni-tuebingen.de/software/locas. Supporting Information Figure S1 Performance comparison of assembly with a sequencing depth of 56 for the first chromosome of A. thaliana. Illumina reads with a sequencing depth of 56 were simulated for the first chromosome of A. thaliana Col-0. The reads were assigned to the reference sequence corresponding to their origin positions and partitioned into blocks of a length of 10 kb. The avgN50 (average N50) is plotted against the avgERR (average error rate) for the assembly tools LOCAS, EULER-SR, ABySS, VELVET and SOAPdenovo. For each assembler, several runs are displayed corresponding to the different parameter settings. The data points of ABySS are drawn in orange, EULER-SR in green, LOCAS in red, VELVET in blue and SOAPdenovo in turquoise. (DOC) Figure S2 Performance comparison of assembly with a sequencing depth of 7.56 for the fourth chromosome of A. thaliana. Illumina reads were simulated for the fourth chromosome of A. thaliana Col-0 with a sequencing depth of 7.56. After the reads were assigned to their origin positions in the reference sequence they were partitioned into blocks of a length of 10 kb. The avgN50 (average N50) is plotted against the avgERR (average error rate) for the assembly tools LOCAS, EULER-SR, ABySS, VELVET and SOAPdenovo. For each assembler, several runs are displayed corresponding to the different parameter settings and ech data point corresponds to such a run. The data points of ABySS are drawn in orange, EULER-SR in green, LOCAS in red, VELVET in blue and SOAPdenovo in turquoise. (DOC) Figure S3 Construction of the overlap graph. Eight aligned reads are displayed on the left. Each read has an alignment to all other reads. Most alignments are overlap alignments, while the alignments among the reads 1 to 3 and 4 to 7 are global alignments. The alignment graph for the aligned reads is shown on the right. For each read, a vertex is introduced into the graph; for each overlap alignment, an overlap edge is LOCAS - A Low Coverage Assembler for Resequencing introduced; for each global alignment, a containment edge. Reads 1 and 4 are chosen as exemplar vertices. For all other reads, all edges are deleted except those leading to the exemplars. The deleted edges are shown as dashed lines; solid lines indicate the edges that have been retained. (DOC) Figure S4 Transitive reduction of the overlap graph. The edge (v, z) is transitive since two edges (v, w) and (w, z) exist and their weights less than or equal to the weight of (v, z). A transitive edge is then reduced. (DOC) Figure S5 Different cycle types in the path graph. (A) An example of an undirected cycle is shown. If the consensus sequences of both cycle branches are similar, the structure arises from a sequencing error or an SNP. If the consensus sequences of both branches are dissimilar a false alignment could be the reason. (B) A directed cycle that usually arises from repeats is shown. (C) A directed cycle that is connected with an undirected cycle is shown. Such a structure can arise from several repeat copies. (DOC) Figure S6 Cutting a directed cycle in the path graph. (A) A directed cycle with short dangling paths is displayed. (B) The cycle is shown after all short paths have been cut off and the cycle has been cut open at each branch vertex. (DOC) Table S1 Evaluation results of low sequencing depth assembly with LOCAS, VELVET, EULER-SR and ABySS. (DOC) Table S2 Evaluation of homology-guided assembly on simulated data with SUPERLOCAS and VELVET. (DOC) Table S3 Evaluation of homology-guided assembly on real world data with LOCAS and VELVET (without utilizing left-over reads). (DOC) Table S4 Evaluation of homology-guided assembly on real world data with SUPERLOCAS and VELVET (utilizing left-over reads). (DOC) Text S1 Supplementary Results and Methods. Results of further assemblies for the first chromosome of A. thaliana at a sequencing depth of 56 and for the fourth chromosome of A. thaliana at a sequencing depth of 76 are shown. In addition, the usage of LOCAS and SUPERLOCAS is described. In the Section Methods the read simulation method, the simulation of a homology-guided assembly and the assembly analysis are described more precisely. (DOC) Acknowledgments We thank David Weese and Knut Reinert for their support with the SeqAn library, Christa Lanz and Jun Cao for their help with sequencing, and Felix Ott for updating the METASIM error model. Author Contributions Conceived and designed the experiments: JK SO KS DH. Performed the experiments: JK SO. Analyzed the data: JK. Contributed reagents/ materials/analysis tools: DW SO KS JK. Wrote the paper: JK DH SO KS. Designed and implemented the software used: JK DH. Contributed (algorithmic) ideas for software: JK DH KS DW SO. PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23455