Monitoring non-small cell lung cancer progression and treatment response through hyaluronic acid in sputum.

We evaluated whether hyaluronan (HA) levels in the sputum could be used as a noninvasive tool to predict progressive disease and treatment response, as detected in a computed tomography scan in non-small cell lung cancer (NSCLC) patients. Sputum samples were collected from 84 patients with histological confirmation of NSCLC, 33 of which were in early-stage and 51 in advanced-stage disease. Patients received systemic chemotherapy (CT) after surgery (n=36), combined CT and immunotherapy (IO) (n=15), or targeted therapy for driver mutation and disease relapse (N=4). The primary end-point was to compare sputum HA levels in two different concentrations of hypertonic saline solution with overall survival (OS) and the secondary and exploratory end-points were radiologic responses to treatment and patient outcome. Higher concentrations of HA in the sputum were significantly associated to factors related to tumor stage, phenotype, response to treatment, and outcome. In the early stage, patients with lower sputum HA levels before treatment achieved a complete tumor response after systemic CT with better progression-free survival (PFS) than those with high HA levels. We also examined the importance of the sputum HA concentration and tumor response in the 51 patients who developed metastatic disease and received CT+IO. Patients with low levels of sputum HA showed a complete tumor response in the computed tomography scan and stable disease after CT+IO treatment, as well as a better PFS than those receiving CT alone. HA levels in sputum of NSCLC patients may serve as a candidate biomarker to detect progressive disease and monitor treatment response in computed tomography scans.

Monitoring non-small cell lung cancer progression and treatment response through hyaluronic acid in sputum

We evaluated whether hyaluronan (HA) levels in the sputum could be used as a noninvasive tool to predict progressive disease and treatment response, as detected in a computed tomography scan in non-small cell lung cancer (NSCLC) patients. Sputum samples were collected from 84 patients with histological confirmation of NSCLC, 33 of which were in early-stage and 51 in advanced-stage disease. Patients received systemic chemotherapy (CT) after surgery (n=36), combined CT and immunotherapy (IO) (n=15), or targeted therapy for driver mutation and disease relapse (N=4). The primary end-point was to compare sputum HA levels in two different concentrations of hypertonic saline solution with overall survival (OS) and the secondary and exploratory end-points were radiologic responses to treatment and patient outcome. Higher concentrations of HA in the sputum were significantly associated to factors related to tumor stage, phenotype, response to treatment, and outcome. In the early stage, patients with lower sputum HA levels before treatment achieved a complete tumor response after systemic CT with better progression-free survival (PFS) than those with high HA levels. We also examined the importance of the sputum HA concentration and tumor response in the 51 patients who developed metastatic disease and received CT+IO. Patients with low levels of sputum HA showed a complete tumor response in the computed tomography scan and stable disease after CT+IO treatment, as well as a better PFS than those receiving CT alone. HA levels in sputum of NSCLC patients may serve as a candidate biomarker to detect progressive disease and monitor treatment response in computed tomography scans.

DNA Mismatch Repair-Deficient Colorectal Carcinoma: Referral Rate for Genetic Cancer Risk Assessment in a Brazilian Cancer Center.

BACKGROUND: Approximately 15% of colorectal cancers (CRCs) are deficient in DNA mismatch repair proteins (dMMR), a characteristic that can occur in both sporadic and hereditary CRC. Due to sparse studies on dMMR CRC in the Brazilian population, we conducted a retrospective analysis of referral rates for Genetic Cancer Risk Assessment of this population and also describing clinical and molecular characterization of these tumors. METHODS: A retrospective, longitudinal, and unicenter study that included patients with dMMR CRC detected by IHC analysis from Pathology Database of our institution, from January 2015 to July 2017. RESULTS: MMR IHC testing was performed in 998 CRC tumors, and 78 tumors (7.8%) had dMMR. The mean age at diagnosis was 56.8 years (17-90), and most patients were female (41 out of 78, 52.6%). Of the 52 patients with right-sided CRC, 40 tumors (77%) had loss of the MLH1 and/or PMS2 expression, and 12 tumors (23%) had loss of MSH2 and/or MSH6 expression (p = 0.005). From 78 patients with dMMR CRC, only 43 patients (55.1%) were referred for genetic counseling (GC), and of them, only 33 patients (76.7%) really went to GC consultation. A total of 21 patients with dMMR CRC performed genetic testing. CONCLUSION: Overall, genetic referral was less than expected in our population. Most of dMMR CRC patients did not receive GC, even in a cancer center, either due to the absence of referral or personal decision and few patients who pursued genetic counseling performed genetic testing.

Contribution of the GSTP1 c.313A>G variant to hearing loss risk in patients exposed to platin chemotherapy during childhood.

BACKGROUND AND AIM: Ototoxicity is a potential adverse effect of chemotherapy with platin drugs, such as cisplatin and carboplatin, in children. Hearing loss (HL) affecting frequencies below 4 kHz can compromise speech perception. The aim of this study was to investigate whether genetic variants previously implicated in ototoxicity are associated with HL overall and HL below 4 kHz in pediatric oncology patients treated with cisplatin or carboplatin. MATERIALS AND METHODS: Patients given cisplatin or carboplatin for a pediatric cancer at least 5 years prior to the start of the study were enrolled. The patients underwent comprehensive audiological evaluations and genotyping to detect the presence of the GJB2 c.35delG, GSTP1 c.313A>G, and MT-RNR1 m.1555A>G polymorphisms. RESULTS: HL was identified in 31/61 patients (50.8%), including 28/42 treated with cisplatin (66.6%) and 3/19 treated with carboplatin (15.8%). HL was associated with higher mean doses of cisplatin (p = .002) and carboplatin (p = .010). The c.313A>G variant of GSTP1 (heterozygous or homozygous) was detected in 31/61 patients (50.8%). An association between this variant allele and HL involving frequencies ≤ 4 kHz was identified (p = .020; 10-fold vs. non-carriers). No associations with HL were observed for GJB2 or MT-RNR1 gene variants. CONCLUSION: The GSTP1 c.313A>G variant may increase the risk of low-frequency HL in pediatric oncology patients treated with cisplatin or carboplatin chemotherapy.

Use of uracil-DNA glycosylase enzyme to reduce DNA-related artifacts from formalin-fixed and paraffin-embedded tissues in diagnostic routine

Background: Detection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine. Methods: We selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples. Results: All UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF < 10%) which are eliminated with UDG treatment. Conclusion: Including UDG enzymatic treatment before multiplex amplification in the NGS workflow significantly decreased the number of artifactual variants detected in FFPE samples. Thus, including this additional step in the current methodology should improve the rate of true mutation detection in the molecular diagnostic routine.

Tissue alkalosis in cold-ischemia time.

The control of pre-analytical-factors in human biospecimens collected for health research is currently required. Only two previous reports using post-mortem brain samples have tried to address the impact of cold-ischemia on tissue pH. Here we report pH variations according to time (third-order polynomial model) in mice for liver, kidney and lung samples. Tissue alkalosis in cold-ischemia time may be an underlying mechanism of gene expression changes. Therefore, tissue-pH regulation after organ removal may minimize biological stress in human tissue samples.

Genomic imbalances pinpoint potential oncogenes and tumor suppressors in Wilms tumors.

BACKGROUND: Wilms tumor (WT) has a not completely elucidated pathogenesis. DNA copy number alterations (CNAs) are common in cancer, and often define key pathogenic events. The aim of this work was to investigate CNAs in order to disclose new candidate genes for Wilms tumorigenesis. RESULTS: Array-CGH of 50 primary WTs without pre-chemotherapy revealed a few recurrent CNAs not previously reported, such as 7q and 20q gains, and 7p loss. Genomic amplifications were exclusively detected in 3 cases of WTs that later relapsed, which also exhibited an increased frequency of gains affecting a 16.2 Mb 1q21.1-q23.2 region, losses at 11p, 11q distal, and 16q, and WT1 deletions. Conversely, aneuploidies of chromosomes 13 and 19 were found only in WTs without further relapse. The 1q21.1-q23.2 gain associated with WT relapse harbours genes such as CHD1L, CRABP2, GJA8, MEX3A and MLLT11 that were found to be over-expressed in WTs. In addition, down-regulation of genes encompassed by focal deletions highlighted new potential tumor suppressors such as CNKSR1, MAN1C1, PAQR7 (1p36), TWIST1, SOSTDC1 (7p14.1-p12.2), BBOX and FIBIN (11p13), and PLCG2 (16q). CONCLUSION: This study confirmed the presence of CNAs previously related to WT and characterized new CNAs found only in few cases. The later were found in higher frequency in relapsed cases, suggesting that they could be associated with WT progression.

Tissue hyaluronan expression, as reflected in the sputum of lung cancer patients, is an indicator of malignancy

Hyaluronan (HA) shows promise for detecting cancerous change in pleural effusion and urine. However, there is uncertainty about the localization of HA in tumor tissue and its relationship with different histological types and other components of the extracellular matrix, such as angiogenesis. We evaluated the association between HA and degree of malignancy through expression in lung tumor tissue and sputum. Tumoral tissue had significantly increased HA compared to normal tissue. Strong HA staining intensity associated with cancer cells was significant in squamous cell carcinoma compared to adenocarcinoma and large cell carcinoma. A significant direct association was found between tumors with a high percentage of HA and MVD (microvessel density) in tumoral stroma. Similarly significant was the direct association between N1 tumors and high levels of HA in cancer cells. Cox multivariate analysis showed significant association between better survival and low HA. HA increased in sputum from lung cancer patients compared to cancer-free and healthy volunteers and a significant correlation was found between HA in sputum and HA in cancer tissue. Localization of HA in tumor tissue was related to malignancy and reflected in sputum, making this an emerging factor for an important diagnostic procedure in patients suspected to have lung cancer. Further study in additional patients in a randomized prospective trial is required to finalize these results and to validate our quantitative assessment of HA, as well as to couple it to gold standard sputum cytology.

Tissue hyaluronan expression, as reflected in the sputum of lung cancer patients, is an indicator of malignancy.

Hyaluronan (HA) shows promise for detecting cancerous change in pleural effusion and urine. However, there is uncertainty about the localization of HA in tumor tissue and its relationship with different histological types and other components of the extracellular matrix, such as angiogenesis. We evaluated the association between HA and degree of malignancy through expression in lung tumor tissue and sputum. Tumoral tissue had significantly increased HA compared to normal tissue. Strong HA staining intensity associated with cancer cells was significant in squamous cell carcinoma compared to adenocarcinoma and large cell carcinoma. A significant direct association was found between tumors with a high percentage of HA and MVD (microvessel density) in tumoral stroma. Similarly significant was the direct association between N1 tumors and high levels of HA in cancer cells. Cox multivariate analysis showed significant association between better survival and low HA. HA increased in sputum from lung cancer patients compared to cancer-free and healthy volunteers and a significant correlation was found between HA in sputum and HA in cancer tissue. Localization of HA in tumor tissue was related to malignancy and reflected in sputum, making this an emerging factor for an important diagnostic procedure in patients suspected to have lung cancer. Further study in additional patients in a randomized prospective trial is required to finalize these results and to validate our quantitative assessment of HA, as well as to couple it to gold standard sputum cytology.

Intratumoral heterogeneity of ADAM23 promotes tumor growth and metastasis through LGI4 and nitric oxide signals.

Intratumoral heterogeneity (ITH) represents an obstacle for cancer diagnosis and treatment, but little is known about its functional role in cancer progression. The A Desintegrin And Metalloproteinase 23 (ADAM23) gene is epigenetically silenced in different types of tumors, and silencing is often associated with advanced disease and metastasis. Here, we show that invasive breast tumors exhibit significant ADAM23-ITH and that this heterogeneity is critical for tumor growth and metastasis. We demonstrate that while loss of ADAM23 expression enhances invasion, it causes a severe proliferative deficiency and is not itself sufficient to trigger metastasis. Rather, we observed that, in ADAM23-heterotypic environments, ADAM23-negative cells promote tumor growth and metastasis by enhancing the proliferation and invasion of adjacent A23-positive cells through the production of LGI4 (Leucine-rich Glioma Inactivated 4) and nitric oxide (NO). Ablation of LGI4 and NO in A23-negative cells significantly attenuates A23-positive cell proliferation and invasion. Our work denotes a driving role of ADAM23-ITH during disease progression, shifting the malignant phenotype from the cellular to the tissue level. Our findings also provide insights for therapeutic intervention, enforcing the need to ascertain ITH to improve cancer diagnosis and therapy.

Papel da proteína Mx1 na resposta a interferons e no processo neoplásico / Role of Mx1 protein in the response to interferons and neoplastic process

A proteína Mx1 é codificada por um gene induzido por interferon e compartilha a organização de seus domínios, a capacidade de homo-oligomerização e associação com membranas com as grandes dinaminas GTPases. A proteína Mx1 está envolvida na resposta contra um grande número de vírus de RNA, como aqueles pertencentes à família Buniavírus e o vírus influenza. Curiosamente, o gene MX1 foi encontrado como silenciado por metilação em diversos processos neoplásicos, incluindo carcinomas de cabeça e pescoço de células escamosas. Neste cenário, o silenciamento gênico de MX1 está associado à imortalização de uma série de linhagens celulares neoplásicas. Assim, Mx1 se destaca como uma das principais proteínas envolvidas nas respostas imunes induzidas por interferon e também desempenha um importante papel no controle do ciclo celular. Aqui discutimos os aspectos funcionais da proteína Mx1 abordando sua atividade antiviral, organização estrutural, envolvimento com neoplasias e, principalmente, os aspectos funcionais obtidos pela determinação de seus parceiros celulares.

The Mx1 protein is encoded by an interferon-induced gene and shares domain organization, homo-oligomerization capacity and membrane association with the large dynamin-like GTPases. The Mx1 protein is involved in the response to a large number of RNA viruses, such as the bunyavirus family and the influenza virus. Interestingly, it has also been found as a methylation-silenced gene in several types of neoplasm, including head and neck squamous cell carcinoma. In this scenario, MX1 gene silencing is associated with immortalization in several neoplastic cell lines. Thus, Mx1 stands out as one of the key proteins involved in interferon-induced immune response and also plays an important role in cell cycle control. Here we discuss some of the functions of the Mx1 protein, including its antiviral activity, protein folding and involvement in neoplasia, as well as those revealed by investigating its cellular partners.

Temporal blastemal cell gene expression analysis in the kidney reveals new Wnt and related signaling pathway genes to be essential for Wilms' tumor onset.

Wilms' tumors (WTs) originate from metanephric blastema cells that are unable to complete differentiation, resulting in triphasic tumors composed of epithelial, stromal and blastemal cells, with the latter harboring molecular characteristics similar to those of the earliest kidney development stages. Precise regulation of Wnt and related signaling pathways has been shown to be crucial for correct kidney differentiation. In this study, the gene expression profile of Wnt and related pathways was assessed in laser-microdissected blastemal cells in WTs and differentiated kidneys, in human and in four temporal kidney differentiation stages (i.e. E15.5, E17.5, P1.5 and P7.5) in mice, using an orthologous cDNA microarray platform. A signaling pathway-based gene signature was shared between cells of WT and of earliest kidney differentiation stages, revealing genes involved in the interruption of blastemal cell differentiation in WT. Reverse transcription-quantitative PCR showed high robustness of the microarray data demonstrating 75 and 56% agreement in the initial and independent sample sets, respectively. The protein expression of CRABP2, IGF2, GRK7, TESK1, HDGF, WNT5B, FZD2 and TIMP3 was characterized in WTs and in a panel of human fetal kidneys displaying remarkable aspects of differentiation, which was recapitulated in the tumor. Taken together, this study reveals new genes candidate for triggering WT onset and for therapeutic treatment targets.

Heteroduplex formation and S1 digestion for mapping alternative splicing sites.

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pairing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks the opposite DNA strand. In order to establish a strategy for mapping alternative splice-prone sites in the whole transcriptome, we developed a method combining the formation of heteroduplexes between 2 distinct splicing variants and S1 nuclease digestion. For 20 consensuses identified here using this methodology, 5 revealed a conserved splice site after inspection of the cDNA alignment against the human genome (exact splice sites). For 8 other consensuses, conserved splice sites were mapped at 2 to 30 bp from the border, called proximal splice sites; for the other 7 consensuses, conserved splice sites were mapped at 40 to 800 bp, called distal splice sites. These latter cases showed a nonspecific activity of S1 nuclease in digesting double-strand DNA. From the 20 consensuses identified here, 5 were selected for reverse transcription-polymerase chain reaction validation, confirming the splice sites. These data showed the potential of the strategy in mapping splice sites. However, the lack of specificity of the S1 nuclease enzyme is a significant obstacle that impedes the use of this strategy in large-scale studies.

Molecular profiling of isolated histological components of Wilms tumor implicates a commom role for the Wnt signaling pathway in kidney and tumor development

Wilms tumor (WT), a tumor composed of three histological components - blastema (BL), epithelia and stroma - is considered an appropriate model system to study the biological relationship between differentiation and tumorigenesis. To investigate molecular associations between nephrogenesis and WT, the gene expression pattern of individual cellular components was analyzed, using a customized platform containing 4,608 genes. WT gene expression patterns were compared to genes regulated during kidney differentiation. BL had a closer gene expression pattern to the earliest stage of normal renal development. The BL gene expression pattern was compared to that of fetal kidney (FK) and also between FK and mature kidney, identifying 25 common deregulated genes supposedly involved in the earliest events of WT onset. Quantitative RT-PCR was performed, confirming the difference in expression levels for 13 of 16 genes (81.2%) in the initial set and 8 of 13 (61.5%) in an independent set of samples. An overrepresentation of genes belonging to the Wnt signaling pathway was identified, namely PLCG2, ROCK2 and adenomatous polyposis coli (APC). Activation of the Wnt pathway was confirmed in WT, using APC at protein level and PLCG2 at mRNA and protein level. APC showed positive nuclear immunostaining for an independent set of WT samples, similarly to the FK in week 11. Lack of PLCG2 expression was confirmed in WT and in FK until week 18. Taken together, these results provided molecular evidence of the recapitulation of the embryonic kidney by WT as well as involvement of the Wnt pathway in the earliest events of WT onset.

Gene expression profiling of clinical stages II and III breast cancer.

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Gene expression profiling of clinical stages II and III breast cancer

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Mining ORESTES no-match database: can we still contribute to cancer transcriptome?

The Human Cancer Genome Project generated about 1 million expressed sequence tags by the ORESTES method, principally with the aim of obtaining data from cancer. Of this total, 341,680 showed no similarity with sequences in the public transcript databases, referred to as [quot ]no-match[quot ]. Some of them represent low abundance or difficult to detect human transcripts, but part of these sequences represent genomic contamination or immature mRNA. We performed a bioinformatics pipeline to determine the novelty of ORESTES [quot ]no-match[quot ] datasets from prostate or breast tissues. We started with 14,908 clusters mapped on the human genome. A total of 2226 clusters originating from more than two libraries or singletons with gaps upon genome alignment were selected. Ninety-four clusters with canonical splice sites representing the most stringent criteria to be considered a gene were subjected to manual inspection regarding genomic hits. Of the manually inspected clusters, 49.6% contained new sequences where 42.2% were probable low-expression alternative forms of the characterized genes and 7.4% unpredicted genes. RT-PCR followed by sequencing was performed to validate the largest spliced sequence from 8 clusters, resulting in the confirmation of five sequences as true human transcript fragments. Some of them were differentially expressed between tumor and normal tissue by an in silico analysis. We can conclude that after clean up of the no-match dataset, we still have about 939 new exons and 165 unpredicted genes that could complete the prostate or breast transcriptome.

Closure of rRNA related gaps in the Chromobacterium violaceum genome with the PCR-assisted contig extension (PACE) protocol

In the finishing phase of the Chromobacterium violaceum genome project, the shotgun sequences were assembled into 57 contigs that were then organized into 19 scaffolds, using the information from shotgun and cosmid clones. Among the 38 ends resulting from the 19 scaffolds, 10 ended with sequences corresponding to rRNA genes (seven ended with the 5S rRNA gene and three ended with the 16S rRNA gene). The 28 non-ribosomal ends were extended using the PCR-assisted contig extension (PACE) methodology, which immediately closed 15 real gaps. We then applied PACE to the 16S rRNA gene containing ends, resulting in eight different sequences that were correctly assembled within the C. violaceum genome by combinatory PCR strategy, with primers derived from the non-repetitive genomic region flanking the 16S and 5S rRNA gene. An oriented combinatory PCR was used to correctly position the two versions (copy A and copy B, which differ by the presence or absence of a 100-bp insert); it revealed six copies corresponding to copy A, and two to copy B. We estimate that the use of PACE, followed by combinatory PCR, accelerated the finishing phase of the C. violaceum genome project by at least 40 per cent

PCR-assisted contig extension: stepwise strategy for bacterial genome closure.

Finishing is rate limiting for genome projects, and improvements in the efficiency of complete genome-sequence compilation will require improved protocols for gap closure. Here we report a novel approach for extending shotgun contigs and closing gaps that we termed PCR-assisted contig extension (PACE). PACE depends on the capture of rare mismatched interactions that occur between arbitrary primers and template DNA of unknown sequence, even under highly stringent conditions, by means of elevated PCR-cycle repetition and the use of specific anchoring primers corresponding to adjacent regions of known sequence. Using PACE, we have generated extensions with an average of 1 kb from all contigs generated from the shotgun sequencing of a 5-Mb genome, which closed the majority of gaps with a single round of experimentation. This included the generation of multiple extensions for contigs that terminated in one of the eight copies of the rRNA operon. We calculate that the switch from shotgun sequencing to PACE should occur between 5- and 8-fold genome coverage for maximum benefit and minimum overall cost. PACE is a robust and straightforward strategy that should simplify the finishing phase of bacterial genome projects.