Clone Pooling Methods for Physical Mapping

产品说明：本提案的主要目标是开发和验证用于BAC文库的快速物理映射的基于池的新方法。第一种方法是短标签合并基因组索引（“ST-PGI”），用于将一个物种的BAC比较物理映射到另一个物种的组装基因组上。第二种方法是用于BAC之间的直接（非比较性）重叠检测的克隆阵列合并猎枪映射（“CAPS-MAP”）方法。 合并基因组索引（“PGI”）是一种用于BAC比较物理图谱的新方法。PGI是通过汇集阵列BAC克隆，生成适当覆盖的鸟枪序列池，并通过使用此信息将单个BAC映射到相关生物体的同源序列上来进行的。前两个步骤，BAC克隆池和鸟枪测序，借鉴了贝勒医学院人类基因组测序中心现有的高通量方法。 最后一步，将池解卷积为特定的BAC，通过将来自池的DNA序列读数与相关生物体的参考基因组序列进行比较来完成。如果来自行池的一个读段和来自列池的一个读段在近距离处与参考序列匹配，则它们都被分配（去卷积）到两个池的交叉处的BAC，并且BAC被映射到两个匹配之间的参考序列的区域上。当定位密切相关的基因组时，通过对包含50至150 bp序列标签的串联体进行测序，可以将PGI的效率提高4至12倍，这种方法我们称为短标签PGI （“ST-PGI”）。 克隆阵列合并猎枪映射（“CAPS-MAP”）是一种与PGI相关的方法。虽然CAPS-MAP和PGI都利用基本上相同的合并实验数据，但CAPS-MAP通过将读段组装成重叠群来解卷积。包含来自行和列的读段的重叠群在交叉点处解卷积为BAC。重叠群解卷积为多个BAC表明BAC重叠。因此，CAPS-MAP提供了一种直接的、非比较性的映射方法，与PGI互补。 该提案旨在进一步开发ST-PGI和CAPS-MAP，并通过实验和模拟验证基于池的多千兆碱基基因组物理映射的综合策略。

DESCRIPTION: (provided by applicant) The main goal of this proposal is to develop and validate novel pooling-based methods for the rapid physical mapping of BAC libraries. The first method is Short-Tag Pooled Genomic Indexing ("ST-PGI") for comparative physical mapping of BACs of one species onto an assembled genome of another. The second method is Clone-Array Pooled Shotgun MAPping ("CAPS-MAP") method for direct (non-comparative) overlap detection between BACs. Pooled Genomic Indexing ("PGI") is a novel method for comparative physical mapping of BACs. PGI is carried out by pooling arrayed BAC clones, generating shotgun sequence pools to appropriate coverage, and by using this information to map individual BACs onto homologous sequences of a related organism. The first two steps, pooling of BAC clones and shotgun sequencing, draw on existing high-throughput methodology at the Baylor College of Medicine Human Genome Sequencing Center. The last step, deconvolution of the pools into specific BACs, is accomplished by comparison of DNA sequence reads from pools against a reference genomic sequence of a related organism. If one read from a row pool and one from a column pool match the reference sequence at a close distance, they are both assigned (deconvoluted) to the BAC at the intersection of the two pools and the BAC is mapped onto the region of the reference sequence between the two matches. When mapping closely related genomes, efficiency of PGI can be increased 4 to l2-fold by sequencing concatenamers comprising 50 to l50 bp sequence tags, a method which we refer to as Short-Tag PGI ("ST-PGI"). Clone-Arrayed Pooled Shotgun MAPping ("CAPS-MAP") is a method related to PGI. While both CAPS-MAP and PGI utilize essentially the same pooling experimental data, CAPS-MAP deconvolutes by assembling reads into contigs. A contig containing a read from a row and a column is deconvoluted to the BAC at the intersection. A contig deconvoluting to multiple BACs indicates BAC overlap. CAPS-MAP thus provides a direct, non-comparative mapping method complementary to PGI. This proposal aims to further develop ST-PGI and CAPS-MAP, and to validate experimentally and by simulation an integrated strategy for pooling-based physical mapping of multi-gigabase genomes.