Biomedical subjects

Genomics

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PubMed1995

Pre-annealing of total genomic DNA probes for simultaneous genomic in situ hybridization.

We used pre-annealing of differently labelled total genomic DNA probes to perform simultaneous genomic in situ hybridization on mitotic and meiotic chromosomes of interspecific hybrids between plant species of the Tribe Triticeae. The species origin of chromosomes was demonstrated by a two-colour fluorescence after in situ hybridization with directly labelled probes incorporating fluorescein (visualized green) and rhodamine (visualized red). The pre-annealing blocked out common DNA sequences between the different genomes, hence increasing species specificity of the probes. The method is simple and rapid because the hybridization takes only about 2 h, including the pre-annealing step, and hence the whole process can be accomplished easily within a working day making it suitable for routine analysis of chromosomes and genomes.

Chromosomes↗

PubMed1986

Human cardiac myosin heavy chain genes. Isolation of a genomic DNA clone and its characterization and of a second unique clone also present in the human genome.

A gene sequence coding for myosin heavy chain (MHC) of human cardiac muscle was isolated by screening a human genomic library with a 32P-labelled 1.1kb SacI restriction fragment from a previously characterized cDNA clone specifying the light meromyosin and 3' untranslated region of mRNA encoding rabbit cardiac alpha-MHC. The DNA of this human genomic clone (lambda HCMHC8) hybridized much more strongly than did other clones isolated under similar, low stringency conditions both to the rabbit cDNA probe and to mRNA isolated from rat cardiac, but not skeletal, muscle tissue. Probe made from a DNA restriction fragment of lambda HCMHC8 hybridized a single 31S band of human ventricular mRNA. This size is identical to that of cardiac MHC mRNA of other species. Heteroduplex analysis showed hybridization of lambda HCMHC8 with exon segments in a rabbit cardiac MHC genomic clone (lambda MHC alpha 12/1). It also showed that lambda HCMHC8 spanned 14 kb of DNA and contained exon segments estimated to code for two-thirds of a MHC including the carboxylic acid terminus. By rescreening the library under more stringent conditions, where only DNA sequences having strong homology to cardiac MHC genes would be expected to hybridize, clones having restriction maps overlapping lambda HCMHC8 were isolated together with a unique clone (lambda HCMHC9). DNA gel blot hybridization of human genomic DNA with lambda HCMHC8 probe at medium stringency gave a pattern of restriction fragments similar to the restriction map of lambda HCMHC8. A weaker set of bands also appeared which corresponded in pattern to the map of lambda HCMHC9.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2004

Genome-wide array comparative genomic hybridization analysis reveals distinct amplifications in osteosarcoma.

BACKGROUND: Osteosarcoma is a highly malignant bone neoplasm of children and young adults. It is characterized by extremely complex karyotypes and high frequency of chromosomal amplifications. Currently, only the histological response (degree of necrosis) to therapy represent gold standard for predicting the outcome in a patient with non-metastatic osteosarcoma at the time of definitive surgery. Patients with lower degree of necrosis have a higher risk of relapse and poor outcome even after chemotherapy and complete resection of the primary tumor. Therefore, a better understanding of the underlying molecular genetic events leading to tumor initiation and progression could result in the identification of potential diagnostic and therapeutic targets. METHODS: We used a genome-wide screening method - array based comparative genomic hybridization (array-CGH) to identify DNA copy number changes in 48 patients with osteosarcoma. We applied fluorescence in situ hybridization (FISH) to validate some of amplified clones in this study. RESULTS: Clones showing gains (79%) were more frequent than losses (66%). High-level amplifications and homozygous deletions constitute 28.6% and 3.8% of tumor genome respectively. High-level amplifications were present in 238 clones, of which about 37% of them showed recurrent amplification. Most frequently amplified clones were mapped to 1p36.32 (PRDM16), 6p21.1 (CDC5L, HSPCB, NFKBIE), 8q24, 12q14.3 (IFNG), 16p13 (MGRN1), and 17p11.2 (PMP22 MYCD, SOX1,ELAC27). We validated some of the amplified clones by FISH from 6p12-p21, 8q23-q24, and 17p11.2 amplicons. Homozygous deletions were noted for 32 clones and only 7 clones showed in more than one case. These 7 clones were mapped to 1q25.1 (4 cases), 3p14.1 (4 cases), 13q12.2 (2 cases), 4p15.1 (2 cases), 6q12 (2 cases), 6q12 (2 cases) and 6q16.3 (2 cases). CONCLUSIONS: This study clearly demonstrates the utility of array CGH in defining high-resolution DNA copy number changes and refining amplifications. The resolution of array CGH technology combined with human genome database suggested the possible target genes present in the gained or lost clones.

Adolescent↗

PubMed2005

High resolution genomic analysis of sporadic breast cancer using array-based comparative genomic hybridization.

INTRODUCTION: Genomic aberrations in the form of subchromosomal DNA copy number changes are a hallmark of epithelial cancers, including breast cancer. The goal of the present study was to analyze such aberrations in breast cancer at high resolution. METHODS: We employed high-resolution array comparative genomic hybridization with 4,134 bacterial artificial chromosomes that cover the genome at 0.9 megabase resolution to analyze 47 primary breast tumors and 18 breast cancer cell lines. RESULTS: Common amplicons included 8q24.3 (amplified in 79% of tumors, with 5/47 exhibiting high level amplification), 1q32.1 and 16p13.3 (amplified in 66% and 57% of tumors, respectively). Moreover, we found several positive correlations between specific amplicons from different chromosomes, suggesting the existence of cooperating genetic loci. Queried by gene, the most frequently amplified kinase was PTK2 (79% of tumors), whereas the most frequently lost kinase was PTK2B (hemizygous loss in 34% of tumors). Amplification of ERBB2 as measured by comparative genomic hybridization (CGH) correlated closely with ERBB2 DNA and RNA levels measured by quantitative PCR as well as with ERBB2 protein levels. The overall frequency of recurrent losses was lower, with no region lost in more than 50% of tumors; the most frequently lost tumor suppressor gene was RB1 (hemizygous loss in 26% of tumors). Finally, we find that specific copy number changes in cell lines closely mimicked those in primary tumors, with an overall Pearson correlation coefficient of 0.843 for gains and 0.734 for losses. CONCLUSION: High resolution CGH analysis of breast cancer reveals several regions where DNA copy number is commonly gained or lost, that non-random correlations between specific amplicons exist, and that specific genetic alterations are maintained in breast cancer cell lines despite repeat passage in tissue culture. These observations suggest that genes within these regions are critical to the malignant phenotype and may thus serve as future therapeutic targets.

Breast Neoplasms↗

PubMed2004

Compendium of genome-wide scans of lipid-related phenotypes: adding a new genome-wide search of apolipoprotein levels.

The genetic dissection of complex inherited diseases is a major challenge. Despite limited success in finding genes, substantial data based on genome-wide scan strategies is now available for a variety of diseases and related phenotypes. This can perhaps best be appreciated in the field of lipid and lipoprotein levels, where the amount of information generated is becoming overwhelming. We have created a database containing the results from whole-genome scans of lipid-related phenotypes undertaken to date. The usefulness of this database is demonstrated by performing a new autosomal genomic scan on apolipoprotein B (apoB), LDL-apoB, and apoA-I levels, measured in 679 subjects of 243 nuclear families. Linkage was tested using both allele-sharing and variance-component methods. Only two loci provided support for linkage with both methods: a LDL-apoB locus on 18q21.32 and an apoA-I locus on 3p25.2. Adding those findings to the database highlighted the fact that the former is reported as a lipid-related locus for the first time, whereas the latter has been observed before. However, concerns arise when displaying all data on the same map, because a large portion of the genome is now covered with loci supported by at least suggestive evidence of linkage.

Adult↗

PubMed2004

Detrended fluctuation analysis of genome-wide copy number profiles of glioblastomas using array-based comparative genomic hybridization.

We examined whole genomic aberrations of biopsied samples from 19 independent glioblastomas by array-based comparative genomic hybridization analysis. The highest frequencies of copy number gains were observed on RFC2 (73.3%), EGFR (63.2%), and FGR, ELN, CDKN1C , FES, TOP2A, and ARSA (57.9% each). The highest frequencies of copy number losses were detected on TBR1 (52.6%), BMI1 (52.6%), EGR2 (47.4%), DMBT1 (47.4%), MTAP (42.1%), and FGFR2 (42.1%). The copy number gains of CDKN1C and INS and the copy number losses of TBR1 were significantly correlated with longer survival of patients. High-level amplifications were identified on EGFR, SAS/CDK4, PDGFRA, MDM2, and ARSA. These genes are assumed to be involved in tumorigenesis or progression of glioblastomas. The first attempts to apply detrended fluctuation analysis to copy number profiles by considering the reading direction as the time axis demonstrated that higher long-term fractal scaling exponents (alpha2) correlated well with longer survival of glioblastoma patients. The present study indicates that array-based comparative genomic hybridization analysis has great potential for assessment of copy number changes and altered chromosomal regions of brain tumors. Furthermore, we show that nonlinear analysis methods of whole genome copy number profiles may provide prognostic information about glioblastoma patients.

Adult↗

PubMed1988

The difference in tumorigenicity between Epstein-Barr virus (EBV) genome-positive and genome-negative epithelial hybrid cell lines derived from the human nasopharynx.

An epithelial/hybrid cell line was previously established (A2L/AH) by the fusion of 8-azaguanine-resistant epithelial cells (Ad-AH) with Epstein-Barr virus (EBV)-transformed lymphoblastoid cells (A2L), as a nasopharyngeal carcinoma (NPC) model cell line in vitro. However, the A2L/AH cells were nontumorigenic in nude mice during early passage levels. In this study, an EBV genome-negative clone and two EBV genome-positive clones of the A2L/AH cells have been successfully cloned. We have continued to culture these three clones, as it is possible that EBV may disturb mechanisms that regulate tumorigenicity when cells are grown in vitro for long periods of time. Two EBV genome-positive A2L/AH clones have become tumorigenic in nude mice after long periods in culture, whereas EBV genome-negative A2L/AH cells were nontumorigenic after the same period of time. The data suggest that EBV may be associated with tumorigenicity in nude mice, at least in part.

Animals↗

PubMed2004

Detection of oncogene amplifications in medulloblastomas by comparative genomic hybridization and array-based comparative genomic hybridization.

OBJECT: Few studies have been conducted to investigate the genomic survey of oncogene amplification in medulloblastoma. Low frequency of N-myc, C-myc, and epidermal grow factor receptor (EGFR) gene amplification (< 10%) has been reported in medulloblastoma. Previous comparative genomic hybridization (CGH) study of primary medulloblastomas has revealed chromosomal amplification on 2p21, 3p, 5p15.3, 7q, 8q24, 11q22.3, and 17q. The aim of this study was to detect common oncogenes involved in medulloblastoma tumorigenesis. METHODS: The authors studied a series of 14 samples by performing CGH and array-based CGH. The CGH analysis detected nonrandom losses on 8p, 17p, 16q, 8q, and 1p, whereas gains were found on 17q, 12q, 7q, and 1p. Array-based CGH was conducted to investigate amplification of 58 oncogenes throughout the genome of these samples. Gene amplifications identified for the first time included PGY1 at 7q21.1, MDM2 at 12q14.3-q15, and ERBB2 at 17q21.2. The highest frequencies of oncogene gain were detected in D17S1670 (61.5%), PIK3CA (46.2%), PGY1 (38.5%), MET (38.5%), ERBB2 (38.5%), and CSE1L (38.5%). The gain in gene copy numbers was confirmed in 34 additional archival medulloblastoma cases by using fluorescence in situ hybridization analysis. CONCLUSIONS: This is the first genome-wide survey of multiple oncogene amplifications involved in the development of medulloblastoma. Gains of several candidate oncogenes such as D17S1670, ERBB2, PIK3CA, PGY1, MET, and CSE1L were frequently detected. These genes may be used as molecular markers and therapeutic targets of medulloblastomas.

Adolescent↗

PubMed1999

Prognostic value of genomic alterations in invasive cervical squamous cell carcinoma of clinical stage IB detected by comparative genomic hybridization.

The clinical behavior of invasive cervical carcinoma of clinical stage IB varies considerably in tumors presenting without regional lymph node metastases. The early identification of patients at higher risk for poor outcome may prove useful because these patients would benefit from aggressive adjuvant treatments. In this study, comparative genomic hybridization was applied to evaluate whether genomic aberrations have prognostic significance in cervical carcinoma. Genomic alterations were evaluated in 62 cervical carcinomas of clinical stage IB. DNA sequence losses were most prevalent at chromosomes 4q (53%), 3p (52%), 13q (45%), 4p (44%), Xq (44%), 5q (40%), 18q (37%), and 6q (35%). Several genomic alterations were associated with poor clinical outcome or metastasis. The total number of DNA aberrations/tumor (P < 0.02) and the number of DNA sequence losses/tumor (P < 0.04) were associated with disease-specific survival. 9p deletions were significantly more frequent in carcinomas with lymph node metastasis than in node-negative tumors (P < 0.03). Losses of chromosome 11p (P < 0.0001) and 18q (P < 0.01) were associated with poor prognosis in cervical carcinomas without lymph node metastasis. These data suggest that inactivation of tumor suppressor genes on chromosomes 9p, 11p, and 18q may play a role in the progression of cervical carcinoma.

Carcinoma, Squamous Cell↗

PubMed2002

Genome wide detection of oncogene amplifications in nasopharyngeal carcinoma by array based comparative genomic hybridization.

We have applied the method of genomic microarray to investigate amplification of oncogenes throughout the genome of nasopharyngeal carcinoma (NPC). Array based comparative genomic hybridization (array CGH) allows simultaneous examination of 58 oncogenes commonly amplified in various human cancers. In the present study, we have examined 15 NPC samples including five cell lines, two xenografts and eight primary tumours with array CGH to reveal the particular oncogenes associated with this cancer. This is the first genome wide survey of multiple oncogene amplifications involved in the development of NPC. Non-random gene amplifications were identified for the first time in NPC on MYCL1 in 1p34.3 and on TERC and PIK3CA at 3q26.3. Other high level amplified oncogenes included NRAS, RAF1, MYB, EGFR, FGF4, EMS1, and D17S167. Highest frequencies of gain of novel oncogenes were detected on MYCL1 (66.7%), TERC (46.7%), ESR (46.7%), PIK3CA (40%), LAMC2 (33.3%), and CSE1L (33.3%).

Carcinoma↗

PubMedDate not supplied

Genome aberrations observed by restriction landmark genome scanning analysis of chromosomal DNA in various types of primary hepatocellular carcinoma.

BACKGROUND/AIMS: The genes responsible for hepatocellular carcinoma have not been identified. To identify the relevant genes of hepatocellular carcinoma, detailed and comprehensive information of genomic aberrations must be obtained. To reveal the chromosomal aberrations in hepatocellular carcinoma, we carried out a restriction landmark genome scanning analysis of various types of hepatocellular carcinoma. METHODOLOGY: Samples of various types of hepatocellular carcinoma, including two with multinodular-hepatocellular carcinomas, one hepatocellular carcinoma showing nodules in a nodule pattern, one hepatocellular carcinoma metastasized to different tissues, three small (< 2.0 cm) hepatocellular carcinomas and four large (> 5.0 cm) hepatocellular carcinomas were examined by the restriction landmark genome scanning method with corresponding non-hepatocellular carcinoma tissues. Restriction enzyme Not I was used as a landmark enzyme, Eco RV was used as a fragmentation enzyme, and Hin fI was used as a digestion enzyme in the gel for two-dimensional electrophoresis. RESULTS: We observed spot aberrations with different origins. Frequently observed spot aberrations originated from the change in the methylation status of repetitive sequences. No clear correlation between the pathological grade and the number or type of spot aberrations was observed. CONCLUSIONS: We demonstrated that major aberrations of restriction landmark genome scanning spots originated from the change of methylation status in hepatocellular carcinoma.

Carcinoma, Hepatocellular↗

PubMed1999

Sequencing of a 3.3 kb Fragment from LsNPV Genome and Comparison of Its Genome Structure with Other Two NPVs.

A fragment (3 336 bp) of LsNPV genome has been sequenced. It contains three complete ORFs (ORF2, ORF3, ORF4) and two incomplete ORFs (ORF1, ORF5). Comparing with AcNPV genome it was found that ORF1 coding product was homologous with P94 protein ORF3, ORF4 and ORF5 were homologous with ORF60, ORF59 and ORF57 of AcNPV respectively. Their transcriptional orientations are identical. It is suggested that the ORF2 is a new gene of LsNPV genome. The results showed that the genomic structure of LsNPV is quite different with that of other baculovirus compared. This may be helpful for investigation of the LsNPV molecular biology.

Journal Article↗

PubMedDate not supplied

Chromosome arm 20q gains and other genomic alterations in esophageal squamous cell carcinoma, as analyzed by comparative genomic hybridization and fluorescence in situ hybridization.

BACKGROUND/AIMS: Our recent analysis of gastric cancers and colorectal cancers using comparative genomic hybridization revealed a novel, high frequent copy number increases the long arm of chromosome 20 in association with possible involvement of liver metastases and poor prognosis. This led to further comparative genomic hybridization analysis of chromosomal aberrations in primary tumors of esophageal squamous cell carcinoma. The aim of the study presented here was to analyze the chromosomal aberrations and to determine the numbers of copies of AIB1, BTAK, DcR3 and E2F1 as putative target genes on chromosome 20q as well as their expression and relation to clinicopathological features in 41 primary tumors of esophageal squamous cell carcinoma. METHODOLOGY: We used comparative genomic hybridization to screen 41 primary tumors of esophageal squamous cell carcinoma for changes in the number of copies of DNA sequences. To further characterize the gain of DNA sequences at 20q, we also performed fluorescence in situ hybridization analysis. We examined the relationship between these changes and clinicopathological factors. RESULTS: Gains in chromosome arm 20q were detected (34.1%) as well as a high level of gain in 20q12-13 (4.8%). AIB1 amplification was observed in 4.9% (2/41), BTAK amplification in 9.8% (4/41), DcR3 amplification was in 4.9% (2/41), and E2F1 amplification in 7.3% (3/41). The survival of patients with BTAK or E2F1 amplification was significantly lower than that of patients without these abnormalities. CONCLUSIONS: These findings provide evidence for a number of previously unknown genomic aberrations in esophageal squamous cell carcinoma, suggesting the existence of target regions relevant to its progression. Esophageal squamous cell carcinoma with 20q gain showed extensive lung metastases, pleural effusion and liver metastases and poorer prognosis compared to cases without 20q gain. Our results suggest that amplification of BTAK or E2F1 are likely to lead to an increase in the number of malignant phenotypes of esophageal squamous cell carcinoma and that these aberrations can be expected to be useful as markers of poor prognosis.

Aged↗

PubMedDate not supplied

[Genome of the pogonophore Siboglinum fiordicum: organizational characteristics and divergence with respect to the genomes of representatives of certain types of invertebrates].

DNA of Pogonophora Siboglinum fiordicum was studied. DNA was isolated from ethanol fixed animals. It contains 40.5% GC according to the melting temperature and 39.3% GC as estimated by cation-exchange chromatography. From the reassociation kinetic data with a [125I] tracer 400 nucleotide long it is clear that 70% of the genome consists of repetitive sequences. The fast component (C0t less than 0.04) constitutes 11% of the genome, the intermediate (C0t 1/2 = 1.5, repetition frequency 1600)--59%. The unique sequences comprise about 30% of the genome. Experiments where [125I]tracers of increasing length were reassociated with driver DNA fragments 300 nucleotides long point out that at least 80% of the genome contains repetitive sequences, part of them being organized in short-period interspersion patterns. The hybridization of Pogonophora [125I]DNA fragments with DNA of invertebrates of other types--Echinodermata, Ascidia, Mollusca, Annelida--did not show any measurable homology between their DNAs neither in repetitive, nor in the unique fraction.

Animals↗

PubMed1996

Gains, losses, and amplification of genomic material in rhabdomyosarcoma analyzed by comparative genomic hybridization.

In this study, 10 embryonal and 14 alveolar rhabdomyosarcoma (RMS) tumor samples, including 4 cell lines derived from tumors of the alveolar subtype, were analyzed by comparative genomic hybridization. In the embryonal tumors, the gain of whole or most of various chromosomes, notably chromosomes 2 (60% of cases), 13 (60%), 12 (60%), 8 (60%), 7 (50%), 17 (40%), 18 (40%), and 19 (40%), and the loss of chromosomes 16 (40%), 10 (30%), 15 (20%), and 14 (20%) were found. One case showed evidence of genomic amplification at 12q13-15. In contrast, the alveolar tumors and cell lines showed consistent evidence of genomic amplification, with multiple amplicons in some cases. The amplicons were localized to l2q13-15 (50%), 2p24 (36%), 13q14 (14%), l3q32 (14%), 1q36 (14%), 1q21 (7%), and 8q13-21 (7%). Four cases had additional copies of chromosome 17 or l7q. These changes were in addition to the presence of fusion gene transcripts that are associated with translocations specific to alveolar RMS. The results show that distinct patterns of primarily gains of specific chromosomal material are associated with the embryonal subtype of RMS, and that genomic amplification seems to play an important role in the alveolar subtype. Notably, these distinct changes predominantly involved chromosomes 2, 12, and 13 in both subtypes.

Adolescent↗

PubMed1997

Extensive genomic abnormalities in childhood medulloblastoma by comparative genomic hybridization.

We analyzed 27 samples of primary medulloblastoma, using comparative genomic hybridization and a novel statistical approach to evaluate chromosomal regions for significant gain or loss of genomic DNA. An array of nonrandom changes was found in most samples. Two discrete regions of high-level DNA amplification of chromosome bands 5p15.3 and 11q22.3 were observed in 3 of 27 tumors. Nonrandom genomic losses were most frequent in regions on chromosomes 10q (41% of samples), 11 (41%), 16q (37%), 17p (37%), and 8p (33%). Regions of DNA gain most often involved chromosomes 17q (48%) and 7 (44%). These findings suggest a greater degree of genomic imbalance in medulloblastoma than has been recognized previously and highlight chromosomal loci likely to contain oncogenes or tumor suppressor genes that may contribute to the molecular pathogenesis of this tumor.

Adolescent↗

PubMed2007

Targets of genome copy number reduction in primary breast cancers identified by integrative genomics.

The identification of specific oncogenes and tumor suppressor genes in regions of recurrent aneuploidy is a major challenge of molecular cancer research. Using both oligonucleotide single-nucleotide polymorphism and mRNA expression arrays, we integrated genomic and transcriptional information to identify and prioritize candidate cancer genes in regions of increased and decreased chromosomal copy number in a cohort of primary breast cancers. Confirming the validity of this approach, several regions of previously-known copy number (CN) alterations in breast cancer could be successfully reidentified. Focusing on regions of decreased CN, we defined a prioritized list of eighteen candidate genes, which included ARPIN, FBN1, and LZTS1, previously shown to be associated with cancers in breast or other tissue types, and novel genes such as P29, MORF4L1, and TBC1D5. One such gene, the RUNX3 transcription factor, was selected for further study. We show that RUNX3 is present at reduced CNs in proportion to the rest of the tumor genome and that RUNX3 CN reductions can also be observed in a breast cancer series from a different center. Using tissue microarrays, we demonstrate in an independent cohort of over 120 breast tissues that RUNX3 protein is expressed in normal breast epithelium but not fat and stromal tissue, and widely down-regulated in the majority of breast cancers (>85%). In vitro, RUNX3 overexpression suppressed the invasive potential of MDA-MB-231 breast cancer cells in a matrigel assay. Our results demonstrate the utility of integrative genomic approaches to identify novel potential cancer-related genes in primary tumors. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Breast Neoplasms↗

PubMed2007

Paired-end genomic signature tags: a method for the functional analysis of genomes and epigenomes.

Because paired-end genomic signature tags are sequenced-based, they have the potential to become an alternate tool to tiled microarray hybridization as a method for genome-wide localization of transcription factors and other sequence-specific DNA binding proteins. As outlined here the method also can be used for global analysis of DNA methylation. One advantage of this approach is the ability to easily switch between different genome types without having to fabricate a new microarray for each and every DNA type. However, the method does have some disadvantages. Among the most rate-limiting steps of our PE-GST protocol are the need to concatemerize the diTAGs, size fractionate them and then clone them prior to sequencing. This is usually followed by additional steps to amplify and size select for long (> or = 500) concatemer inserts prior to sequencing. These time-consuming steps are important for standard DNA sequencing as they increase efficiency approximately 20-30-fold since each amplified concatemer can now provide information on multiple tags; the limitation on data acqui- sition is read length during sequencing. However, the development of new sequencing methods such as Life Sciences' 454 new nanotechnology-based sequencing instrument (41) could increase tag sequencing efficiency by several orders of magnitude (> or = 100,000 diTAG reads/run), which is sufficient to provide in-depth global analysis of all ChIP PE-GSTs in a single run. This is because the lengths of our paired-end diTAGs (approximately 60 bp) fall well within the region of high accuracy for read lengths on this instrument. In principle, sequence analysis of diTAGs could begin as soon as they are generated, thereby completely bypassing the need for the concatemerization, sizing, downstream cloning steps and sequencing template purification. In addition, our protocol places any one of several unique four-base long nucleotide sequences, such as GATC, between each and every diTAG pair, which could be used to help the instrument's software keep base register and also provide a well-located peak height indicator in the middle of every sequence run. This additional feature could permit multiplexing of the data by simultaneous sequencing of several pooled libraries if each used a different linker sequence during diTAG formation (Figure 4).

Base Sequence↗