Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing

Abstract

Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.

In-depth sequence analysis of the tomato chromosome 12 centromeric region: identification of a large CAA block and characterization of pericentromere retrotranposons

“…After sequence assembly and alignment of the three BAC clones, one physical gap remained between BAC clones 78G17 and 121H12 and was closed using a bridging clone derived from an overlapping BAC 128G09. The bridging clone was fully sequenced using transposonmediated sequencing as described by Yang et al (2003Yang et al ( , 2005.…”

Toward closing rice telomere gaps: mapping and sequence characterization of rice subtelomere regions

“…We screened the OSJNPb and OSJNPc rice genomic libraries, representing 8.8 · genome equivalents, with 166,752, and 138,960 clones, respectively; the average size of each insert was 10.8 kb (10-kb libraries) for clones containing rice telomeric repeats (http:// www.genome.arizona.edu; Yang et al 2003). Eleven high-density colony filters of the two libraries were gridded in a 5·5-array pattern on 22.5·22.5-cm Hybond N+ filters (Amersham, UK) and hybridized with a telomere-specific overgo probe (OVG-A: CCCTAAAC-CCTAAACCCTAAACCC; OVG-B: TAGGGTTTAG-GGTTTAGGGTTTAG).…”

“…Base-calling was performed automatically using PHRED, and vector sequences were removed by CROSS_MATCH ). We applied the TGS system F-700 transposon method (Finnzymes, Espoo, Finland) for complete sequencing of the selected 10-kb clones (Yang et al 2003). High-quality, vector-trimmed sequences were then used for the sequence assembly of the 10-kb clones using PHRAP and CONSED (Gordon et al 1998).…”

“…Nipponbare), we screened the two 10-kb libraries with the (CCCTAAA) 5 telomere repeat (Yang et al 2003) and identified around 200 positive clones, of which 96 were end-sequenced. The ten clones showing telomere repeats at one end were fully sequenced.…”

“…Two HaeIII clones, pb083I20 and pb027O22, fortuitously showed the correct HaeIII site (cc) by starting with cc of the telomere motif. The resulting analyses indicate that most of the distal telomere sequences were illegitimately ligated into the EcoRV-digested blunt end vector for the HaeIII library, whereas, it is likely that a fraction of the cohesive ends of the BamHI-digested cloning vector were damaged during preparation of dephophorylated linear vector (Yang et al 2003;Kim et al 2004) (e.g., mechanical breakage or exonuclease contamination of the restriction enzyme or CIP), resulting in blunt ends that would be suitable for telomere cloning. Distal telomere ends are known to contain a single G overhang.…”