Results

Overview of Project   Mapping Reagents   Integration with Genetic, RH, and Cytogenetic Maps   General Features of Assembled YAC Contigs and STS Maps   Unanchored Contigs   Gaps on Contig Map   Apparent False-Negative and False-Positive Data   Complex Chromosomal Regions   Indices of Map Completion   Relationship Between Genetic and Physical Distance   Data Dissemination

Overview of Project

Since the onset of the Human Genome Project, we have focused on constructing a detailed physical map of human chromosome 7 by a YAC-based STS-content mapping strategy (Green and Green 1991; Green et al. 1991a; Green et al. 1994; Green et al. 1995a; Bouffard et al. 1997). Specifically, our goals included: (1) mapping an STS, on average, every 100 kb across the chromosome [a programmatic goal of the U.S. Human Genome Project (Collins and Galas 1993)] and integration of the YAC-based physical map with the cytogenetic, genetic, and RH maps of the chromosome; (2) construction of a map that covered the great majority of the chromosome, that established an unique order for most of the mapped STSs, and that provided large, contiguous stretches of redundant clone coverage.

Here, we report that these goals have been reached. In brief, our physical map has been assembled to near completion, with the construction of 22 well-positioned YAC contigs that together account for virtually all of the chromosome (Table 1). Supplementing this paper is more detailed information that can be accessed electronically on the World Wide Web (Table 2). Below are descriptions of the key aspects of our maps and the associated reagents. Specifically, we discuss the: (1) important characteristics of the STSs and YACs that constitute the map; (2) major features of the mapping strategy employed; (3) details about the assembled YAC-based physical maps and their integration with other chromosome 7 maps; (4) quality and assessed completion of the map; (5) complex regions of chromosome 7 that are difficult to map; and (6) routes by which our data, maps, and reagents can be accessed.

In constructing the physical map reported here, we have mapped 2150 chromosome 7 STSs (see Methods for details). Importantly, these STSs were intentionally derived from a variety of sources of DNA sequence (Table 3). Similarly, the YACs used for assembling the contig maps represent a diverse set of clones highly enriched for chromosome 7 DNA, including both relatively non-chimeric clones derived from a human-hamster monochromosomal hybrid cell line and clones isolated from total genomic libraries (mostly from the CEPH YAC library; see Methods for details).

Some important characteristics about the YAC collection are evident based on the screens for 2173 chromosome 7 STSs. At least one positive YAC was identified for 2150 (99%) of these STSs (Table 3). There were an average of 9.9 positive YACs per STS and an average of 5.5 STSs in each STS-containing YAC, although these numbers varied for the different sources of YACs (Table 4). Based on analysis of the resulting contigs, it is possible to estimate the relative contribution from each clone source. YACs constructed from the monochromosomal hybrid cell line account for 38-40% of the total clone contribution in the contigs, the CEPH clones 50-52%, and the other YACs 8-12%. Thus, the relative clone contribution within our contig maps is similar, but not identical, for the small but mostly non-chimeric hybrid cell line-derived YACs and the large but often-chimeric CEPH YACs. It should be stressed that at least one positive hybrid cell line-derived YAC has been identified for 95% of the STSs, while at least one positive CEPH YAC has been identified for 90% of the STSs.

As a high priority, we integrated our YAC-based physical map with the chromosome 7 genetic, RH, and cytogenetic maps. Cross-referencing different genomic maps enhances the utility of the maps, confirms the deduced STS order, and orders and orients the evolving YAC contigs (Green et al. 1994; Hudson et al. 1995; Bray-Ward et al. 1996).

Microsatellite-based genetic markers can uniquely serve as landmarks on both genetic and physical maps. The Genethon genetic map of human chromosome 7 consists of 272 (CA)n-repeat polymorphisms positioned across 204 cM (Weissenbach et al. 1992; Gyapay et al. 1994; Dib et al. 1996; see http://www.genethon.fr/genethon_en.html). PCR assays suitable for YAC library screening were successfully developed for 262 of these markers (Green et al. 1994), and in all but two instances, at least one positive YAC was identified.

We also established the YAC contig location of markers comprising a recently-constructed chromosome 7 RH map (Stewart, E.A., K.B. McKusick, A. Aggarwal, S. Brady, G. diSibio, D. Elam, N. Fang, R. Goold, M. Harris, R. Lee, et al., in prep.). This map was derived by analyzing the Stanford G2 whole-genome RH panel (consisting of 85 human-hamster somatic hybrid cell lines) with 268 chromosome 7 STSs, many of which were selected based on their positions on the evolving YAC contigs. These markers mapped to 107 uniquely ordered RH bins (with each bin placed at >1000:1 odds) and were distributed across 5070 cR10,000. PCR assays suitable for YAC library screening were successfully developed for 259 of these markers, and in every instance, at least one positive YAC was identified.

Information derived from three independent mapping methods (YAC-based STS-content, genetic, and RH mapping) was thus considered in constructing the integrated physical map reported here. Figure 1 provides a summary of the total number of chromosome 7 STSs analyzed by each of these methods. Note that 66 STSs were mapped by all three methods, while many others were analyzed by two of the methods. In many cases, different PCR assays were used for localizing an STS on the various maps [e.g., we preferred to design new primers for mapping Genethon genetic markers on the YAC contigs rather than use the ones employed for genotyping (Green et al. 1994)]. Electronic summary tables containing relevant information about the STSs corresponding to the markers on the Genethon genetic map (example shown in Figure 2) and chromosome 7 RH map (example shown in Figure 3) are available on the World Wide Web (Table 2).

For integrating the YAC-based physical map with the cytogenetic map, clones at regular intervals across the assembled contigs were mapped by fluorescence in situ hybridization (FISH) to establish their relative locations on metaphase chromosomes. Particular emphasis was placed on analyzing YACs that contain Genethon genetic markers, thereby allowing integration of the physical, genetic, and cytogenetic maps at numerous points along the chromosome (Green et al. 1994). The electronic summary table (see Figure 2) provides the established cytogenetic positions for those Genethon genetic markers whose corresponding YACs were analyzed by FISH.

The strategy for map construction (described in Methods), which made extensive use of the program SEGMAP, was utilized to localize 2150 chromosome 7 STSs relative to 3892 YACs, resulting in the construction of 36 contigs (Table 1). A representative sample of a SEGMAP-constructed contig map is provided in Figure 4. Overall, the YAC-based STS-content analysis allowed 90% of the mapped STSs to be uniquely ordered within each contig. Furthermore, >95% of adjacent STS pairs are connected by two or more YACs (i.e., double-linked), thereby providing strong evidence for their close proximity on chromosome 7. For each of the 36 YAC contigs, an electronic summary table listing the STSs in their deduced map order and containing relevant information about each STS is available on the World Wide Web (Table 2), with a representative example shown in Figure 5.

Of the 36 contigs, 22 are anchored in place relative to the Genethon genetic, RH, and/or cytogenetic maps (Table 1, Figures 2 and 3) accounting for >98% of the mapped STSs. For the subset of STSs corresponding to markers on the Genethon genetic and chromosome 7 RH maps, the STS order deduced by YAC-based mapping is remarkably consistent with that determined by these alternate methods. However, a small number of inconsistencies remain between the YAC contig map and the Genethon genetic (e.g., sWSS3069, sWSS3046, sWSS1221) and chromosome 7 RH (e.g., sWSS2745, sWSS1090, sWSS1713) maps. Specifically, 17 of 260 (7%) Genethon genetic markers mapped to YAC contigs and 12 of 259 (5%) RH markers mapped to YAC contigs appear to be at discrepant locations relative to their positions established by genetic and RH mapping (at >1000:1 odds), respectively. Four of the RH mapping cases correspond to markers mapping near the centromere, which is not unexpected given the difficulties inherent to the analysis of regions residing near centromeres by RH mapping methods (Cox et al. 1990; James et al. 1994; Walter et al. 1994; Hudson et al. 1995; Schuler et al. 1996; Stewart et al. 1997). All discrepancies with the Genethon genetic and RH maps are indicated in the electronic summary tables (Table 2, Figures 2 and 3).

Each of the 22 anchored YAC contigs has been assigned two names: one given by SEGMAP that reflects the lowest-numbered STS in the contig and one indicating its long-range position relative to the other anchored contigs (A through V, respectively). Within the first and last anchored contigs, A (sWSS1361) and V (sWSS25), are 7p and 7q telomere-containing YACs, respectively. Neither of the telomeric contigs contains STSs corresponding to markers present on the Genethon genetic or RH maps; however, their terminal locations and relative orientations are known by the positions of the telomere-containing YACs. Of the remaining contigs, 18 are anchored on both the Genethon genetic and RH maps, while contig B (sWSS457) is only anchored on the Genethon genetic map and contig S (sWSS806) is only anchored on the RH map. Despite the occasional minor discrepancies with the Genethon genetic or RH maps (e.g., contigs P and R relative to the Genethon genetic map, contigs G, R, and S relative to the RH map), the predicted relative order of the anchored contigs seems reasonable in light of the available long-range mapping information. In addition to establishing the relative order for most of the anchored contigs, the Genethon genetic and RH maps allow determination of the relative orientation for all but a few contigs (Table 1). In total, 19 of the anchored YAC contigs, containing just under 98% of the mapped STSs, are both ordered and oriented. Figure 6 provides a global overview of the predicted relative positioning of the 22 anchored YAC contigs across the chromosome.

The 22 anchored YAC contigs are highly variable in size and complexity (Table 1). The number of STSs in each contig ranges from 2 to 634 (with an average of 96), while the number of YACs range from 5 to 1260 (with an average of 175). In routine usage, SEGMAP depicts contigs so that the STSs are evenly spaced and the size of each YAC, in general, reflects the number of STSs it contains (see Figure 4). However, SEGMAP is capable of accounting for the measured insert size and STS content of each YAC to estimate the relative STS spacing and clone overlaps. The resulting computed maps thus provide estimates of total contig sizes, with the 22 anchored contigs averaging 7.95 Mb in size (Table 1). The largest YAC contig [E (sWSS9)] contains 634 STSs and 1260 YACs, spans an estimated 49.6 Mb, represents most of the short arm of the chromosome, and, to our knowledge, is the largest single YAC contig assembled to date.

While the computed maps provide some insight about STS spacing and overall contig lengths, they are largely based on the measured size of each YAC. This measurement is limited by a number of factors, including the frequent presence of large chimeric segments in YACs [particularly clones from total human genomic libraries (Green et al. 1991b)] that make the cloned insert inappropriately large, the common occurrence of internal deletions in YACs that make the cloned insert inappropriately small, and the inherent inaccuracies associated with the high-throughput sizing of 3892 YACs by pulsed-field gel electrophoresis. Note that the cumulative size of the 36 YAC contigs exceeds 186 Mb, whereas chromosome 7 is estimated to contain ~170 Mb of DNA (Trask et al. 1989; Morton 1991). This discrepancy could reflect the above problems with calculating YAC contig sizes and/or an underestimate for the total size of the chromosome.

Numerous indices can be used for assessing the 'completion' of a physical map. While the various parameters mentioned above provide insight about the map, several other features are important to consider. One such feature is the fraction of the chromosome accounted for by the physical map. For example, 99% of the chromosome 7 STSs were successfully mapped to one of the YAC contigs. Thus, assuming that our collection of STSs accurately represents chromosome 7, then 99% of chromosome 7 is accounted for by the assembled YAC contigs. Another parameter that can be considered is the cumulative size of the YAC contigs (Table 1), which totals ~186 Mb and presumably reflects virtually all of chromosome 7.

For some applications, such as positional cloning (Collins 1995b; Green et al. 1995b), the entry point to the chromosome 7 physical map will be through one or more genetic markers. Thus, we estimated the measured the fraction of the chromosome that is physically connected by YACs to Genethon genetic markers using the total collection of STSs as a reflection of the chromosome (Green et al. 1994) (Table 3). Remarkably, 87% of the chromosome 7 STSs are present on the same YAC(s) as a Genethon genetic marker(s), while an additional 10% are present on the same YAC contig (but not the same YAC) as a Genethon marker(s). Only 3% of our STSs map to a YAC contig devoid of a Genethon genetic marker. These percentages are generally the same for STSs derived from different DNA sources (Table 3).

Another important index of map completion is STS resolution. At the simplest level, mapping 2150 STSs across a ~170-Mb chromosome provides an average STS spacing of ~79 kb. If only the 1933 uniquely ordered STSs are considered, then the map contains an uniquely ordered STS, on average, every 88 kb. More rigorous approaches for calculating resolution that account for the relative distribution of STSs (Olson and Green 1993; Cox et al. 1994) can also be used. For the latter analysis, SEGMAP uses the computed contig maps, with the inter-STS spacing estimated, to calculate the fraction of the chromosome accounted for within set bounds around each STS. Figure 7 depicts the results of analyzing our chromosome 7 physical map in this fashion, as deduced by SEGMAP. Roughly 60% of the chromosome appears to reside within 100 kb of an STS on our map, 84% is within 200 kb, and 94% is within 300 kb.

The availability of a complete physical map of a human chromosome, including the established positions for a large set of genetic markers, provided an unique opportunity to correlate genetic and physical distances across an entire chromosome. For this analysis, the 22 anchored YAC contigs were examined in their computed form, and the physical distance between each successive Genethon genetic marker was established. The resulting data allowed the ratio between genetic and physical distances to be calculated in successive intervals (Figure 8). This analysis revealed several interesting features of recombination across this chromosome, including some that cannot be revealed from the genetic map alone. The ratio of genetic to physical distance varies four-fold across human chromosome 7 and is highest near both telomeres, with a progressive decrease towards the centromere from either telomere. The ratio is lowest near the centromere (at roughly 60 Mb) as well as at an internal region near the middle of 7q (at roughly 130 Mb), in both instances indicating regions of decreased recombination. Whether the variation in recombination across a chromosome is caused by specific DNA sequences per se or the local chromatin structure is currently unknown. However, the specific regions of increased and decreased recombination can be further studied using the map and reagents we have generated.

The data and information associated with the chromosome 7 physical map reported here have been organized for dissemination on the World Wide Web, with the URL addresses for key sites listed in Table 2. The most challenging aspect of this process was establishing a mechanism for disseminating the contig maps. The most effective solution to this problem has been to use the program Chromoscope (Zhang et al. 1994), which provides the ability to display and manipulate highly complex maps imported in ASN.1 format (J. Zhang and J. Ostell, unpublished data) and allows our contigs to be accessed in the Entrez Genomes Division at the National Center for Biotechnology Information (NCBI). A representative example of a Chromoscope-depicted contig map is shown in Figure 4B.