External-loop free energy affects dye-labeled terminators premature terminations in DNA cycle-sequencing reactions

Although dideoxy terminators labeled with energy-transfer dyes (BigDyes) provide the most versatile method of automated DNA sequencing, premature terminations result in a substantially reduced reading length of the DNA sequence. I recently demonstrated that combining the annealing step with the extension step at a single temperature (60°C) reduces premature terminations of DNA sequences that ordinarily contain premature terminations when three temperature steps are used in sequencing. I studied a novel class of DNA sequences of 100-bp length and located upstream from the point that causes premature terminations. I determined the thermodynamics of 49 DNA sequences with premature terminations at three temperature steps by using DNA mfold profiles. Sequencing results for 28 samples were improved with two-step cycle sequencing, whereas two-step cycle-sequencing reactions did not improve the results for 21 sequences. Nearest-neighbor thermodynamic parameters for all 49 sequences were compared at temperatures 50°C and 60°C. The parameters predicted that thermodynamic free-base (external-loop) energies (δδG°) were significantly different for these two study groups of samples. The results indicate that changes in free energy in single-strand base (external-loop) sequences can have a significant effect in reducing premature terminations in DNA sequencing reactions run with energy-transfer-based fluorescent-dye terminators.     

Two-step cycle sequencing reduces premature terminations when using primers with high annealing temperatures

Direct cycle sequencing of double-stranded polymerase chain reaction (PCR) products using thermostable polymerase produces fragments that are shorter than expected when the enzyme prematurely detaches as it approaches the 5′-end of the DNA template. These premature terminations result in a substantially reduced reading length of the DNA sequence. Since some DNA templates spontaneously fold and form stable secondary structures at temperatures that are typically used for primer annealing, one factor that may cause premature terminations to occur is the formation of secondary structures in the template during the annealing step of the cycle sequencing reaction. We describe a simple and effective method for reducing premature terminations in DNA sequences. We demonstrate that maintaining the annealing temperature of the cycle sequencing reaction above a critical temperature reduces premature terminations in DNA sequences that regularly contain premature terminations when the temperature of the annealing step is 60°C. In the method described, annealing and extension of the primer along the template take place at the same temperature (72°C). This procedure for reducing premature terminations can be applied when sequencing with primers that are relatively long (at least 27 mer) and have high optimal annealing temperatures.     

No-wash ethanol precipitation of dye-labeled reaction products improves DNA sequencing reads

The advent of DNA sequencing has significantly accelerated molecular biology and clinical genetic testing. Despite recent increases in next-generation sequencing throughput, the most popular platform for DNA sequencing is still the multi-capillary DNA sequencer, which is ideally suited for small-scale sequencing projects and is highly accurate. However, the methods remain time-consuming and laborious. Here, I describe a modified ethylenediaminetetraacetic acid (EDTA) method that skips the washing step in ethanol precipitation. My improvements to standard methods save labor, time, and cost per run and increase the sequence reads by 5 to 10%. This modified method will provide immediate benefits to many researchers.     

DNA sequencing with solid-phase-capturable dideoxynucleotides and energy transfer primers

False terminations occurring in fluorescent dye-primer DNA sequencing, and nonsequencing primer extension DNA fragments generated in dye-terminator sequencing cause background noise in fluorescent electropherograms, leading to errors in sequence determination. We describe here a DNA sequencing chemistry that produces accurate and clean sequencing data on a fluorescent DNA sequencer, eliminating the false terminations and background noise. The procedure involves coupling fluorescence energy transfer (ET) primers that produce high fluorescent signals with solid-phase-capturable biotinylated dideoxynucleotides to generate Sanger DNA sequencing fragments. After the sequencing reaction,the DNA extension fragments that carry a biotin at the 3' end are captured with streptavidin-coated magnetic beads, while the other components in the sequencing reaction are washed away. Only pure DNA extension products terminated by the biotinylated dideoxynucleotides are released from the magnetic beads and are loaded onto a sequencing gel to produce accurate sequencing data.     

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.     

Recent advances in DNA sequencing methods - general principles of sample preparation

DNA sequencing has revolutionized biomedicine, and progress in the field has been unrelenting since it was invented over 30 years ago. The complete DNA sequence of the human genome was obtained as the culmination of a decade of work by a large number of scientists. Less than ten years later, so-called ‘next-generation’ instruments now make it possible for a single lab to produce the same amount of data in a week. But while the instruments are increasingly automated, upstream sample processing remains a challenge. Here I review the current state of the art in preparing genomic and RNA samples for high throughput sequencing.     

Specific-primer-directed DNA sequencing using automated fluorescence detection.

Automated fluorescence-based DNA sequence analysis offers the possibility to undertake very large scale sequencing projects. Directed strategies, such as the specific-primer-directed sequencing approach ('gene walking'), should prove useful in such projects. Described herein is a study involving the use of this approach in conjunction with automated fluorescence detection on a commercial instrument (ABI 370A DNA sequencer). This includes procedures for the rapid chemical synthesis and purification of labeled primers, the design of primer sequences that are compatible with the commercial analysis software, and automated DNA sequence analysis using such primers. A set of four fluorophore-labeled primers can be reliably synthesized in a twenty-four hour period, and greater than 300 nucleotides of analyzed new sequence obtained using this set in an additional twenty-four hours. Scale-up of these procedures to take advantage of the full capabilities of the sequencer is, at present, too slow and costly to be suitable for routine sequencing, and therefore the use of specific-primers is best suited to the closure of gaps in extended sequence produced using random cloning and sequencing strategies.     

DNA barcodes from century‐old type specimens using next‐generation sequencing

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next‐generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century‐old type specimens of Lepidoptera by attempting to recover 164‐bp and 94‐bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories – high (164‐bp sequence), medium (94‐bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR‐based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.     

DNA sequencing research group: 2006 general survey of DNA sequencing facilities.

Over the past few years, technological advances in automated DNA sequencing have had a profound effect on the nature of DNA sequencing laboratories. To characterize the changes occurring within DNA sequencing facilities, the DNA Sequencing Research Group conducted three previous studies, in 1998, 2000, and 2003. A new general survey has been designed and conducted by the DSRG to capture the current status of DNA sequencing facilities in all sectors. Included were questions regarding facility administration, pricing, instrumentation, technology, protocols, and operation. The results of the survey are presented here, accompanied by comparisons to the previous surveys. These comparisons formed a basis for the discussion of trends within the facilities in response to the dynamics of a changing technology.     

Method enabling fast partial sequencing of cDNA clones

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.     

Denaturing gradient gel electrophoresis (DGGE) screening of clones prior to sequencing

Whilst cloning and sequencing techniques are used ubiquitously for the identification of novel DNA sequences, the necessity to determine a consensus sequence means that this can be both labour‐intensive and costly. Here we describe a rapid and cost effective method of using denaturing gradient gel electrophoresis (DGGE) for the analysis of large numbers of clones prior to sequencing. This procedure allows for the selection of specific clones, eliminates the need to sequence multiple copies of the same clone, and reduces the likelihood of sequencing recombinant PCR product.     

Direct sequencing of tobacco chloroplast genome by the polymerase chain reaction

We have developed a polymerase chain reaction (PCR) method for sequencing of tobacco chloroplast genome. In a mixture containing chloroplast DNA, 5-end-labeled oligonucleotide primer, Taq DNA polymerase and reaction buffer, we were able to sequence a segment of chloroplast 16S rRNA gene. The results showed that the 750 bp of DNA sequenced were identical to the sequence reported, indicating that direct sequencing method that we have developed is useful for the sequencing of chloroplast genome. To analyze the chloroplast genome more rapidly in those in vitro grown plantlets, we also developed a simple method which is applicable for the amplifications and sequencing of chloroplast 16S rRNA fragment from either 0.15 g of tobacco leaf or stem tissue. The readable sequences obtained from the presented methods were consistent with the published sequence.     

Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.     

Design of efficient simplified genomic DNA and bisulfite sequencing in large plant populations

The next generation sequencing enables generation of high resolution and high throughput data for structure sequence of any genome at a fast declining cost. This opens opportunity for population based genetic and genomic analyses. In many applications, whole genome sequencing or re-sequencing is unnecessary or prohibited by budget limits. The Reduced Representation Genome Sequencing (RRGS), which sequences only a small proportion of the genome of interest, has been proposed to deal with the situations. Several forms of RRGS are proposed and implemented in the literature. When applied to plant or crop species, the current RRGS protocols shared a key drawback that a significantly high proportion (up to 60%) of sequence reads to be generated may be of non-genomic origin but attributed to chloroplast DNA or rRNA genes, leaving an exceptional low efficiency of the sequencing experiment. We recommended and discussed here the design of optimized simplified genomic DNA and bisulfite sequencing strategies, which may greatly improves efficiency of the sequencing experiments by bringing down the presentation of the undesirable sequencing reads to less than 10% in the whole sequence reads. The optimized RAD-seq and RRBS-seq methods are potentially useful for sequence variant screening and genotyping in large plant/crop populations.     

Direct haplotype-specific DNA sequencing

Determining haplotype-specific DNA sequence information is very important in a wide range of research fields. However, no simple and robust approaches are currently available for determining haplotype-specific sequence information. We have addressed this problem by developing a very simple and robust haplotype-specific sequencing approach. We utilise the fact that DNA sequencing polymerases are sensitive to 3'end mismatches in the sequencing primer. By using two sequencing primers with 3'end corresponding to the two alleles in a given SNP locus, we are able to obtain allele-specific DNA sequences from both alleles. We evaluated this direct haplotype-specific approach by determining haplotypes within the intron 2 sequence of the fructan-6-fructosyltransferase (6-ft) gene in Lolium perenne L. We obtained reliable haplotype-specific sequences for all primers and genotypes evaluated. We conclude that the haplotype-specific sequencing is robust, and that the approach has a potentially very wide application range for any diploid organism.     

T7 DNA polymerase in automated dideoxy sequencing.

T7 DNA polymerase with chemically inactivated 3'-5' exo-nuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laser-excited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.     

DNA sequencing by denaturation: principle and thermodynamic simulations

We describe a new DNA sequencing method called sequencing by denaturation (SBD). A Sanger dideoxy sequencing reaction is performed on the templates on a solid surface to generate a ladder of DNA fragments randomly terminated by fluorescently labeled dideoxyribonucleotides. The labeled DNA fragments are sequentially denatured from the templates and the process is monitored by measuring the change in fluorescence intensities from the surface. By analyzing the denaturation profiles, the base sequence of the template can be determined. Using thermodynamic principles, we simulated the denaturation profiles of a series of oligonucleotides ranging from 12 to 32 bases and developed a base-calling algorithm to decode the sequences. These simulations demonstrate that DNA molecules up to 20 bases can be sequenced by SBD. Experimental measurements of the melting profiles of DNA fragments in solution confirm that DNA sequences can be determined by SBD. The potential limitations and advantages of SBD are discussed. With SBD, millions of sequencing reactions can be performed on a small area on a surface in parallel with a very small amount of sequencing reagents. Therefore, DNA sequencing by SBD could potentially result in a significant increase in speed and reduction in cost in large-scale genome resequencing.     

Modular tagging of amplicons using a single PCR for high‐throughput sequencing

High‐throughput sequencing (HTS) of PCR amplicons is becoming the method of choice to sequence one or several targeted loci for phylogenetic and DNA barcoding studies. Although the development of HTS has allowed rapid generation of massive amounts of DNA sequence data, preparing amplicons for HTS remains a rate‐limiting step. For example, HTS platforms require platform‐specific adapter sequences to be present at the 5′ and 3′ end of the DNA fragment to be sequenced. In addition, short multiplex identifier (MID) tags are typically added to allow multiple samples to be pooled in a single HTS run. Existing methods to incorporate HTS adapters and MID tags into PCR amplicons are either inefficient, requiring multiple enzymatic reactions and clean‐up steps, or costly when applied to multiple samples or loci (fusion primers). We describe a method to amplify a target locus and add HTS adapters and MID tags via a linker sequence using a single PCR. We demonstrate our approach by generating reference sequence data for two mitochondrial loci (COI and 16S) for a diverse suite of insect taxa. Our approach provides a flexible, cost‐effective and efficient method to prepare amplicons for HTS.     

amplisas: a web server for multilocus genotyping using next‐generation amplicon sequencing data

Next‐generation sequencing (NGS) technologies are revolutionizing the fields of biology and medicine as powerful tools for amplicon sequencing (AS). Using combinations of primers and barcodes, it is possible to sequence targeted genomic regions with deep coverage for hundreds, even thousands, of individuals in a single experiment. This is extremely valuable for the genotyping of gene families in which locus‐specific primers are often difficult to design, such as the major histocompatibility complex (MHC). The utility of AS is, however, limited by the high intrinsic sequencing error rates of NGS technologies and other sources of error such as polymerase amplification or chimera formation. Correcting these errors requires extensive bioinformatic post‐processing of NGS data. Amplicon Sequence Assignment (amplisas ) is a tool that performs analysis of AS results in a simple and efficient way, while offering customization options for advanced users. amplisas is designed as a three‐step pipeline consisting of (i) read demultiplexing, (ii) unique sequence clustering and (iii) erroneous sequence filtering. Allele sequences and frequencies are retrieved in excel spreadsheet format, making them easy to interpret. amplisas performance has been successfully benchmarked against previously published genotyped MHC data sets obtained with various NGS technologies.