Inferring Short-Range Linkage Information from Sequencing Chromatograms

Direct Sanger sequencing of viral genome populations yields multiple ambiguous sequence positions. It is not straightforward to derive linkage information from sequencing chromatograms, which in turn hampers the correct interpretation of the sequence data. We present a method for determining the variants existing in a viral quasispecies in the case of two nearby ambiguous sequence positions by exploiting the effect of sequence context-dependent incorporation of dideoxynucleotides. The computational model was trained on data from sequencing chromatograms of clonal variants and was evaluated on two test sets of in vitro mixtures. The approach achieved high accuracies in identifying the mixture components of 97.4% on a test set in which the positions to be analyzed are only one base apart from each other, and of 84.5% on a test set in which the ambiguous positions are separated by three bases. In silico experiments suggest two major limitations of our approach in terms of accuracy. First, due to a basic limitation of Sanger sequencing, it is not possible to reliably detect minor variants with a relative frequency of no more than 10%. Second, the model cannot distinguish between mixtures of two or four clonal variants, if one of two sets of linear constraints is fulfilled. Furthermore, the approach requires repetitive sequencing of all variants that might be present in the mixture to be analyzed. Nevertheless, the effectiveness of our method on the two in vitro test sets shows that short-range linkage information of two ambiguous sequence positions can be inferred from Sanger sequencing chromatograms without any further assumptions on the mixture composition. Additionally, our model provides new insights into the established and widely used Sanger sequencing technology. The source code of our method is made available at http://bioinf.mpi-inf.mpg.de/publications/beggel/linkageinformation.zip.     

Improving the limit of detection for Sanger sequencing: A comparison of methodologies for KRAS variant detection

Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in which different alleles were expected as heterozygous mixtures of 50%. Increasingly, the requirements for variant detection are an analytic sensitivity for minor alleles of <20%, in particular, when assessing the mutational status of heterogeneous tumor samples. Here, we describe a simple modification to the FTSS workflow that improves the limit of detection of cell-line gDNA mixtures from 50%-20% to 5% for G>A transitions and from 50%-5% to 5% for G>C and G>T transversions. In addition, we use two different sample types to compare the limit of detection of sequence variants in codons 12 and 13 of the KRAS gene between Sanger sequencing and other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), and real-time PCR (qPCR).     

Mitochondrial DNA Variant Discovery and Evaluation in Human Cardiomyopathies through Next-Generation Sequencing

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We “shotgun” sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche''s 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300× average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20× while heteroplasmic variants required >200× coverage. Several Sanger “misses” were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.     

Base-Calling Algorithm with Vocabulary (BCV) Method for Analyzing Population Sequencing Chromatograms

Sanger sequencing is a common method of reading DNA sequences. It is less expensive than high-throughput methods, and it is appropriate for numerous applications including molecular diagnostics. However, sequencing mixtures of similar DNA of pathogens with this method is challenging. This is important because most clinical samples contain such mixtures, rather than pure single strains. The traditional solution is to sequence selected clones of PCR products, a complicated, time-consuming, and expensive procedure. Here, we propose the base-calling with vocabulary (BCV) method that computationally deciphers Sanger chromatograms obtained from mixed DNA samples. The inputs to the BCV algorithm are a chromatogram and a dictionary of sequences that are similar to those we expect to obtain. We apply the base-calling function on a test dataset of chromatograms without ambiguous positions, as well as one with 3–14% sequence degeneracy. Furthermore, we use BCV to assemble a consensus sequence for an HIV genome fragment in a sample containing a mixture of viral DNA variants and to determine the positions of the indels. Finally, we detect drug-resistant Mycobacterium tuberculosis strains carrying frameshift mutations mixed with wild-type bacteria in the pncA gene, and roughly characterize bacterial communities in clinical samples by direct 16S rRNA sequencing.     

The Accuracy,Feasibility and Challenges of Sequencing Short Tandem Repeats Using Next-Generation Sequencing Platforms

To date we have little knowledge of how accurate next-generation sequencing (NGS) technologies are in sequencing repetitive sequences beyond known limitations to accurately sequence homopolymers. Only a handful of previous reports have evaluated the potential of NGS for sequencing short tandem repeats (microsatellites) and no empirical study has compared and evaluated the performance of more than one NGS platform with the same dataset. Here we examined yeast microsatellite variants from both long-read (454-sequencing) and short-read (Illumina) NGS platforms and compared these to data derived through Sanger sequencing. In addition, we investigated any locus-specific biases and differences that might have resulted from variability in microsatellite repeat number, repeat motif or type of mutation. Out of 112 insertion/deletion variants identified among 45 microsatellite amplicons in our study, we found 87.5% agreement between the 454-platform and Sanger sequencing in frequency of variant detection after Benjamini-Hochberg correction for multiple tests. For a subset of 21 microsatellite amplicons derived from Illumina sequencing, the results of short-read platform were highly consistent with the other two platforms, with 100% agreement with 454-sequencing and 93.6% agreement with the Sanger method after Benjamini-Hochberg correction. We found that the microsatellite attributes copy number, repeat motif and type of mutation did not have a significant effect on differences seen between the sequencing platforms. We show that both long-read and short-read NGS platforms can be used to sequence short tandem repeats accurately, which makes it feasible to consider the use of these platforms in high-throughput genotyping. It appears the major requirement for achieving both high accuracy and rare variant detection in microsatellite genotyping is sufficient read depth coverage. This might be a challenge because each platform generates a consistent pattern of non-uniform sequence coverage, which, as our study suggests, may affect some types of tandem repeats more than others.     

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。     

Highly efficient somatic-mutation identification using Escherichia coli mismatch-repair detection

The discovery of somatic mutations in cancer tissue is extremely laborious, time-consuming and costly. In an evaluation comparing mismatch repair detection (MRD) against Sanger sequencing for somatic-mutation detection, we found that MRD had a specificity of 96% and a sensitivity of 92%. Our results showed that MRD is a robust and cost-effective alternative to Sanger sequencing for identifying somatic mutations in human tumors.     

Detection and resolution of Cryptosporidium species and species mixtures by genus-specific nested PCR-restriction fragment length polymorphism analysis, direct sequencing, and cloning

Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.     

Calreticulin Mutations in Myeloproliferative Neoplasms: Comparison of Three Diagnostic Methods

Calreticulin (CALR) mutations have recently been reported in 70–84% of JAK2V617F-negative myeloproliferative neoplasms (MPN), and this detection has become necessary to improve the diagnosis of MPN. In a large single-centre cohort of 298 patients suffering from Essential Thrombocythemia (ET), the JAK2V617F, CALR and MPL mutations were noted in 179 (60%), 56 (18.5%) and 13 (4.5%) respectively. For the detection of the CALR mutations, three methods were compared in parallel: high-resolution melting-curve analysis (HRM), product-sizing analysis and Sanger sequencing. The sensitivity for the HRM, product-sizing analysis and Sanger sequencing was 96.4%, 98.2% and 89.3% respectively, whereas the specificity was 96.3%, 100% and 100%. In our cohort, the product-sizing analysis was the most sensitive method and was the easiest to interpret, while the HRM was sometimes difficult to interpret. In contrast, when large series of samples were tested, HRM provided results more quickly than did the other methods, which required more time. Finally, the sequencing method, which is the reference method, had the lowest sensitivity but can be used to describe the type of mutation precisely. Altogether, our results suggest that in routine laboratory practice, product-sizing analysis is globally similar to HRM for the detection of CALR mutations, and that both may be used as first-line screening tests. If the results are positive, Sanger sequencing can be used to confirm the mutation and to determine its type. Product-sizing analysis provides sensitive and specific results, moreover, with the quantitative measurement of CALR, which might be useful to monitor specific treatments.     

Sequencing of large double-stranded DNA using the dideoxy sequencing technique

The dideoxy sequencing technique has been applied to the direct sequencing of large double-stranded DNA molecules with a small single-stranded primer. For instance, the method was applied to the lambda genome, which contains 48 502 base-pairs (Sanger F, Coulson AR, Hong GF, Hill D & Petersen GB, 1982, J. Mol. Biol., in press), and the coding region for gene W identified. The procedure proves useful in the sequence analysis of a large number of different mutations in a particular region and in the analysis of eukaryotic DNA cloned in plasmids, phages, and cosmids.     

Mass Spectrometry-Based Comparative Sequence Analysis for the Genetic Monitoring of Influenza A(H1N1)pdm09 Virus

The pandemic influenza A (H1N1) 2009 virus (pH1N1) contains novel gene segments of zoonotic origin that lack virulence and antiviral resistance markers. We aimed to evaluate the applicability and accuracy of mass spectrometry-based comparative sequence analysis (MSCSA) to detect genetic mutations associated with increased virulence or antiviral resistance in pH1N1. During the 2009 H1N1 pandemic, routine surveillance specimens and clinical antiviral resistance monitoring specimens were analyzed. Routine surveillance specimens obtained from 70 patients with pH1N1 infection were evaluated for mutations associated with increased virulence (PB1-F2, PB2 and NS1 genes) or antiviral resistance (neuraminidase gene, NA) using MSCSA and Sanger sequencing. MSCSA and Sanger sequencing results revealed a high concordance (nucleotides >99%, SNPs ∼94%). Virulence or resistance markers were not detected in routine surveillance specimens: all identified SNPs encoded for silent mutations or non-relevant amino acid substitutions. In a second study population, the presence of H275Y oseltamivir resistant virus was identified by real-time PCR in 19 of 35 clinical antiviral resistance monitoring specimens obtained from 4 immunocompromised patients with ≥14 days prolonged pH1N1 excretion. MSCSA detected H275Y in 24% (4/19) of positive specimens and Sanger sequencing in 89% (17/19). MSCSA only detected H275Y when the mutation was dominant in the analyzed specimens. In conclusion, MSCSA may be used as a rapid screening tool during molecular surveillance of pH1N1. The low sensitivity for the detection of H275Y mutation in mixed viral populations suggests that MSCSA is not suitable for antiviral resistance monitoring in the clinical setting.     

HITAC-seq enables high-throughput cost-effective sequencing of plasmids and DNA fragments with identity

DNA sequencing is vital for many aspects of biological research and diagnostics. Despite the development of second and third generation sequencing technologies, Sanger sequencing has long been the only choice when required to precisely track each sequenced plasmids or DNA fragments. Here, we report a complete set of novel barcoding and assembling system, Highly-parallel Indexed Tagmentation-reads Assembled Consensus sequencing(HITAC-seq), that could massively sequence and track the identities of each individual sequencing sample. With the cost of much less than that of single read of Sanger sequencing,HITAC-seq can generate high-quality contiguous sequences of up to 10 kilobases or longer. The capability of HITAC-seq was confirmed through large-scale sequencing of thousands of plasmid clones and hundreds of amplicon fragments using approximately 100 pg of input DNAs. Due to its long synthetic length, HITACseq was effective in detecting relatively large structural variations, as demonstrated by the identification of a～1.3 kb Copia retrotransposon insertion in the upstream of a likely maize domestication gene. Besides being a practical alternative to traditional Sanger sequencing, HITAC-seq is suitable for many highthroughput sequencing and genotyping applications.     

Comparison of Major and Minor Viral SNPs Identified through Single Template Sequencing and Pyrosequencing in Acute HIV-1 Infection

Massively parallel sequencing (MPS) technologies, such as 454-pyrosequencing, allow for the identification of variants in sequence populations at lower levels than consensus sequencing and most single-template Sanger sequencing experiments. We sought to determine if the greater depth of population sampling attainable using MPS technology would allow detection of minor variants in HIV founder virus populations very early in infection in instances where Sanger sequencing detects only a single variant. We compared single nucleotide polymorphisms (SNPs) during acute HIV-1 infection from 32 subjects using both single template Sanger and 454-pyrosequencing. Pyrosequences from a median of 2400 viral templates per subject and encompassing 40% of the HIV-1 genome, were compared to a median of five individually amplified near full-length viral genomes sequenced using Sanger technology. There was no difference in the consensus nucleotide sequences over the 3.6kb compared in 84% of the subjects infected with single founders and 33% of subjects infected with multiple founder variants: among the subjects with disagreements, mismatches were found in less than 1% of the sites evaluated (of a total of nearly 117,000 sites across all subjects). The majority of the SNPs observed only in pyrosequences were present at less than 2% of the subject’s viral sequence population. These results demonstrate the utility of the Sanger approach for study of early HIV infection and provide guidance regarding the design, utility and limitations of population sequencing from variable template sources, and emphasize parameters for improving the interpretation of massively parallel sequencing data to address important questions regarding target sequence evolution.     

Next-generation phylogeography: a targeted approach for multilocus sequencing of non-model organisms

The field of phylogeography has long since realized the need and utility of incorporating nuclear DNA (nDNA) sequences into analyses. However, the use of nDNA sequence data, at the population level, has been hindered by technical laboratory difficulty, sequencing costs, and problematic analytical methods dealing with genotypic sequence data, especially in non-model organisms. Here, we present a method utilizing the 454 GS-FLX Titanium pyrosequencing platform with the capacity to simultaneously sequence two species of sea star (Meridiastra calcar and Parvulastra exigua) at five different nDNA loci across 16 different populations of 20 individuals each per species. We compare results from 3 populations with traditional Sanger sequencing based methods, and demonstrate that this next-generation sequencing platform is more time and cost effective and more sensitive to rare variants than Sanger based sequencing. A crucial advantage is that the high coverage of clonally amplified sequences simplifies haplotype determination, even in highly polymorphic species. This targeted next-generation approach can greatly increase the use of nDNA sequence loci in phylogeographic and population genetic studies by mitigating many of the time, cost, and analytical issues associated with highly polymorphic, diploid sequence markers.     

A Next Generation Semiconductor Based Sequencing Approach for the Identification of Meat Species in DNA Mixtures

The identification of the species of origin of meat and meat products is an important issue to prevent and detect frauds that might have economic, ethical and health implications. In this paper we evaluated the potential of the next generation semiconductor based sequencing technology (Ion Torrent Personal Genome Machine) for the identification of DNA from meat species (pig, horse, cattle, sheep, rabbit, chicken, turkey, pheasant, duck, goose and pigeon) as well as from human and rat in DNA mixtures through the sequencing of PCR products obtained from different couples of universal primers that amplify 12S and 16S rRNA mitochondrial DNA genes. Six libraries were produced including PCR products obtained separately from 13 species or from DNA mixtures containing DNA from all species or only avian or only mammalian species at equimolar concentration or at 1:10 or 1:50 ratios for pig and horse DNA. Sequencing obtained a total of 33,294,511 called nucleotides of which 29,109,688 with Q20 (87.43%) in a total of 215,944 reads. Different alignment algorithms were used to assign the species based on sequence data. Error rate calculated after confirmation of the obtained sequences by Sanger sequencing ranged from 0.0003 to 0.02 for the different species. Correlation about the number of reads per species between different libraries was high for mammalian species (0.97) and lower for avian species (0.70). PCR competition limited the efficiency of amplification and sequencing for avian species for some primer pairs. Detection of low level of pig and horse DNA was possible with reads obtained from different primer pairs. The sequencing of the products obtained from different universal PCR primers could be a useful strategy to overcome potential problems of amplification. Based on these results, the Ion Torrent technology can be applied for the identification of meat species in DNA mixtures.     

Analysis of the DNA Sequencing Quality and Efficiency of the Apollo100 Robotic Microcycler in a Core Facility Setting

Sanger, or dideoxynucleotide sequencing, is an important tool for biomolecular research. An important trend in DNA sequencing is to find new and innovative ways to provide high-quality, reliable sequences in a more efficient manner, using automated capillary electrophoresis. The Apollo100 combines Sanger cycle sequencing and solid-phase reversible immobilization for product purification in a single instrument with robotic liquid handling and microfluidic (Microscale On-chip Valve) chips that have onboard thermal cycling and pneumatic mixing. Experiments were performed to determine how the DNA sequencing results from the Apollo100 compared with conventional, manual methods used in a core facility setting. Through rigorous experimentation of multiple baseline runs and a dilution series of template concentration, the Apollo100 generated sequencing that exceeded 900 bases with a quality score of 20 or above. When comparing actual client samples of amplicons, plasmids, and cosmids, Apollo100 sequencing results did not differ significantly from those reactions prepared manually. In addition, bacterial genomic DNA was sequenced successfully, directly with the Apollo100, although results were of lower quality than the standard manual method. As a result of the microscale capabilities, the Apollo100 offers valuable savings with respect to the quantity of reagents consumed compared with current manual sequencing methods, thereby continuing the demand for smaller template and reagent requirements. In conclusion, the Apollo100 can generate high-quality DNA sequences for common templates equivalent to those produced using manual sequencing methods and increases efficiency through reduced labor and reagents.     

Sequencing from compomers: using mass spectrometry for DNA de novo sequencing of 200+ nt.

One of the main endeavors in today's life science remains the efficient sequencing of long DNA molecules. Today, most de novo sequencing of DNA is still performed using the electrophoresis-based Sanger concept of 1977, in spite of certain restrictions of this method. Methods using mass spectrometry to acquire the Sanger sequencing data are limited by short sequencing lengths of 15-25 nt. We propose a new method for DNA sequencing using base-specific cleavage and mass spectrometry that appears to be a promising alternative to classical DNA sequencing approaches. A single stranded DNA or RNA molecule is cleaved by a base-specific (bio-)chemical reaction using, for example, RNAses. The cleavage reaction is modified such that not all, but only a certain percentage of bases are cleaved. The resulting mixture of fragments is then analyzed using MALDI-TOF mass spectrometry, whereby we acquire the molecular masses of fragments. For every peak in the mass spectrum, we calculate those base compositions that will potentially create a peak of the observed mass and, repeating the cleavage reaction for all four bases, finally try to uniquely reconstruct the underlying sequence from these observed spectra. This leads us to the combinatorial problem of sequencing from compomers and, finally, to the graph-theoretical problem of finding a walk in a subgraph of the de Bruijn graph. Application of this method to simulated data indicates that it might be capable of sequencing DNA molecules with 200+ nt.     

丙型肝炎病毒准种血清学检测技术的建立

目的:建立一种以血清学为基础的丙型肝炎病毒(HCV)准种检测技术。方法:自20份HCV血清中各挑选30个克隆进行测序比较,分析HCV准种的复杂程度;以HCV准种代表性抗原组合制备免疫芯片,用血清学检测技术分析上述20份HCV血清中的准种变异程度;比较两种方法之间的检出灵敏度和相关性。结果:测序法检出灵敏度为70.0%,血清学检测法检出灵敏度为95.0%,后者显著高于前者(P0.05);两种方法检测结果的相关性为74.7%(P0.01)。结论:血清学检测技术操作简单,且能够反映丙型肝炎患者的HCV准种变异程度,适于临床推广。     

Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations

Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice-COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice-COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice-COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice-COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics.     

Draft genome sequence of Streptomyces clavuligerus NRRL 3585, a producer of diverse secondary metabolites

Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.