Ultra-high resolution array painting facilitates breakpoint sequencing.

OBJECTIVE: To describe a considerably advanced method of array painting, which allows the rapid, ultra-high resolution mapping of translocation breakpoints such that rearrangement junction fragments can be amplified directly and sequenced. METHOD: Ultra-high resolution array painting involves the hybridisation of probes generated by the amplification of small numbers of flow-sorted derivative chromosomes to oligonucleotide arrays designed to tile breakpoint regions at extremely high resolution. RESULTS AND DISCUSSION: How ultra-high resolution array painting of four balanced translocation cases rapidly and efficiently maps breakpoints to a point where junction fragments can be amplified easily and sequenced is demonstrated. With this new development, breakpoints can be mapped using just two array experiments: the first using whole-genome array painting to tiling resolution large insert clone arrays, the second using ultra-high-resolution oligonucleotide arrays targeted to the breakpoint regions. In this way, breakpoints can be mapped and then sequenced in a few weeks.

The complex nature of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes.

OBJECTIVE: To describe the systematic analysis of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes, characterise the structural chromosome rearrangements, map the translocation breakpoints, and report detectable genomic imbalances. METHODS: DNA microarrays were used with a resolution of 1 Mb for the detailed genome-wide analysis of the patients. Array CGH was used to screen for genomic imbalance and array painting to map chromosome breakpoints rapidly. These two methods facilitate rapid analysis of translocation breakpoints and screening for cryptic chromosome imbalance. Breakpoints of rearrangements were further refined (to the level of spanning clones) using fluorescence in situ hybridisation where appropriate. RESULTS: Unexpected additional complexity or genome imbalance was found in six of 10 patients studied. The patients could be grouped according to the general nature of the karyotype rearrangement as follows: (A) three cases with complex multiple rearrangements including deletions, inversions, and insertions at or near one or both breakpoints; (B) three cases in which, while the translocations appeared to be balanced, microarray analysis identified previously unrecognised imbalance on chromosomes unrelated to the translocation; (C) four cases in which the translocation breakpoints appeared simple and balanced at the resolution used. CONCLUSIONS: This high level of unexpected rearrangement complexity, if generally confirmed in the study of further patients, will have an impact on current diagnostic investigations of this type and provides an argument for the more widespread adoption of microarray analysis or other high resolution genome-wide screens for chromosome imbalance and rearrangement.

The DNA sequence and comparative analysis of human chromosome 10.

The finished sequence of human chromosome 10 comprises a total of 131,666,441 base pairs. It represents 99.4% of the euchromatic DNA and includes one megabase of heterochromatic sequence within the pericentromeric region of the short and long arm of the chromosome. Sequence annotation revealed 1,357 genes, of which 816 are protein coding, and 430 are pseudogenes. We observed widespread occurrence of overlapping coding genes (either strand) and identified 67 antisense transcripts. Our analysis suggests that both inter- and intrachromosomal segmental duplications have impacted on the gene count on chromosome 10. Multispecies comparative analysis indicated that we can readily annotate the protein-coding genes with current resources. We estimate that over 95% of all coding exons were identified in this study. Assessment of single base changes between the human chromosome 10 and chimpanzee sequence revealed nonsense mutations in only 21 coding genes with respect to the human sequence.

The DNA sequence and analysis of human chromosome 13.

Chromosome 13 is the largest acrocentric human chromosome. It carries genes involved in cancer including the breast cancer type 2 (BRCA2) and retinoblastoma (RB1) genes, is frequently rearranged in B-cell chronic lymphocytic leukaemia, and contains the DAOA locus associated with bipolar disorder and schizophrenia. We describe completion and analysis of 95.5 megabases (Mb) of sequence from chromosome 13, which contains 633 genes and 296 pseudogenes. We estimate that more than 95.4% of the protein-coding genes of this chromosome have been identified, on the basis of comparison with other vertebrate genome sequences. Additionally, 105 putative non-coding RNA genes were found. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) among human chromosomes, and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb.

Applications of combined DNA microarray and chromosome sorting technologies.

The sequencing of the human genome has led to the availability of an extensive mapped clone resource that is ideal for the construction of DNA microarrays. These genomic clone microarrays have largely been used for comparative genomic hybridisation studies of tumours to enable accurate measurement of copy number changes (array-CGH) at increased resolution. We have utilised these microarrays as the target for chromosome painting and reverse chromosome painting to provide a similar improvement in analysis resolution for these studies in a process we have termed array painting. In array painting, chromosomes are flow sorted, fluorescently labelled and hybridised to the microarray. The complete composition and the breakpoints of aberrant chromosomes can be analysed at high resolution in this way with a considerable reduction in time, effort and cytogenetic expertise required for conventional analysis using fluorescence in situ hybridisation. In a similar way, the resolution of cross-species chromosome painting can be improved and we present preliminary observations of the organisation of homologous DNA blocks between the white cheeked gibbon chromosome 14 and human chromosomes 2 and 17.

The DNA sequence and analysis of human chromosome 6.

Chromosome 6 is a metacentric chromosome that constitutes about 6% of the human genome. The finished sequence comprises 166,880,988 base pairs, representing the largest chromosome sequenced so far. The entire sequence has been subjected to high-quality manual annotation, resulting in the evidence-supported identification of 1,557 genes and 633 pseudogenes. Here we report that at least 96% of the protein-coding genes have been identified, as assessed by multi-species comparative sequence analysis, and provide evidence for the presence of further, otherwise unsupported exons/genes. Among these are genes directly implicated in cancer, schizophrenia, autoimmunity and many other diseases. Chromosome 6 harbours the largest transfer RNA gene cluster in the genome; we show that this cluster co-localizes with a region of high transcriptional activity. Within the essential immune loci of the major histocompatibility complex, we find HLA-B to be the most polymorphic gene on chromosome 6 and in the human genome.

Array painting: a method for the rapid analysis of aberrant chromosomes using DNA microarrays.

OBJECTIVE: The authors describe a method, termed array painting, which allows the rapid, high resolution analysis of the content and breakpoints of aberrant chromosomes. METHODS: Array painting is similar in concept to reverse chromosome painting and involves the hybridisation of probes generated by PCR of small numbers of flow sorted chromosomes on large insert genomic clone DNA microarrays. RESULTS: and CONCLUSIONS: By analysing patients with cytogenetically balanced chromosome rearrangements, the authors show the effectiveness of array painting as a method to map breakpoints prior to cloning and sequencing chromosome rearrangements.

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X.

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.