At the contrary some others are based on relatively low sequence coverage, with several unresolved gaps.
Three unresolved gaps remain in the physical map (see additional data file 1, blau_map.pdf).
As a first step, data from previously sequenced BACs that span genes MELO3C004258 (partially) to MELO3C004290 (partially) was analyzed to close unresolved gaps.
First, re-sequencing by Illumina paired-end reads was used to close gaps and correct false SNPs and small indels; secondly, a PCR experimental approach was used to close still unresolved gaps, particularly those affecting ORFs.
The procedure to close unresolved gaps and correct any remaining sequence errors detected is described in detail in the Methods section and included the use of previously available BAC sequence information [ 14, 15, 17, 18], PCR-aided gap closing, and manual correction of the final sequence by visually inspecting the mapping-alignments of the re-sequencing reads.
The results we present suggest PacBio data will be of immense utility for the development of genome sequence assemblies containing fewer unresolved gaps and ambiguities and a significantly smaller number of contigs than could be produced using short-read sequence data alone.
Recent research on the adverse health effects of fine particle pollution shows estimated concentration-response functions that are approximately linear, with no evidence of safe threshold levels; moreover, unresolved gaps in understanding exist concerning who is most at risk or most susceptible [ 10].
Combined, these factors often make it impossible to span repetitive regions, which therefore collapse into single consensus contigs during sequence assemblies and leave unresolved sequence gaps.
The MELO3C004235-MELO3C004331 region was further analyzed in search of still unresolved sequence gaps, and pairs of primers were designed flanking the selected regions using the Primer3 software in an attempt to close gaps by PCR [ 31].
Before their reassembly with AlignGraph, we reduced the number of unresolved sequence regions (gaps filled with ambiguous N bases) in the scaffolds by applying the GapFiller algorithm, which is currently one of the most efficient gap-filling methods (Boetzer and Pirovano, 2012).
