Heparin sequencing

Heparin is a highly sulfated glycosaminoglycan widely used as an anticoagulant. Modifications in its relatively uniform structure appear to be key to its recognition and modulation of serine proteases, growth factors, chemokines, and extracellular proteins, as has been most clearly demonstrated in the antithrombin binding site. We sequenced the major oligosaccharides released from mastocytoma heparin by partial nitrous acid using a highly sensitive technique tailored for sequencing of metabolically radiolabeled heparin. It utilizes partial nitrous acid cleavage to allow simultaneous sequencing of the internal components of the oligosaccharide under investigation by specific lysosomal exoenzymes. Sequencing revealed that although the majority of the heparin disaccharides are N-, 2-O-, and 6-O-sulfated, the less sulfated disaccharides (lacking 2-O- or 6-O-sulfates) seem to be spaced out along the chain. The technique may be particularly useful for characterizing heparin from novel sources, such as the glial progenitor cells and Ascidia, as well as for sequencing protein binding sites.     

Evaluation of next generation sequencing platforms for population targeted sequencing studies

Background

Next generation sequencing (NGS) platforms are currently being utilized for targeted sequencing of candidate genes or genomic intervals to perform sequence-based association studies. To evaluate these platforms for this application, we analyzed human sequence generated by the Roche 454, Illumina GA, and the ABI SOLiD technologies for the same 260 kb in four individuals.

Results

Local sequence characteristics contribute to systematic variability in sequence coverage (>100-fold difference in per-base coverage), resulting in patterns for each NGS technology that are highly correlated between samples. A comparison of the base calls to 88 kb of overlapping ABI 3730xL Sanger sequence generated for the same samples showed that the NGS platforms all have high sensitivity, identifying >95% of variant sites. At high coverage, depth base calling errors are systematic, resulting from local sequence contexts; as the coverage is lowered additional 'random sampling' errors in base calling occur.

Conclusions

Our study provides important insights into systematic biases and data variability that need to be considered when utilizing NGS platforms for population targeted sequencing studies.     

Generations of sequencing technologies

Advancements in the field of DNA sequencing are changing the scientific horizon and promising an era of personalized medicine for elevated human health. Although platforms are improving at the rate of Moore's Law, thereby reducing the sequencing costs by a factor of two or three each year, we find ourselves at a point in history where individual genomes are starting to appear but where the cost is still too high for routine sequencing of whole genomes. These needs will be met by miniaturized and parallelized platforms that allow a lower sample and template consumption thereby increasing speed and reducing costs. Current massively parallel, state-of-the-art systems are providing significantly improved throughput over Sanger systems and future single-molecule approaches will continue the exponential improvements in the field.     

ABI sequencing analysis

The ABI Sequencing Analysis application is designed specifically for the analysis of data produced by the ABI DNA Sequencer. The ABI sequencer is a laser-based instrument that utilizes fluorescent labels to analyze the products of a sequencing reaction as they migrate through a gel. After the data are collected from a sequencing run, the Analysis program identifies and tracks the sample lanes of the gel and subsequently normalizes and integrates the raw data into a chromatogram of the final sequence. For the use, there are basically two types of files that can be manipulated to potentially improve the analysis results. The Gel File consists of a computer generated image of the sequencing gel with the fluorescent DNA banding patterns. This image allows the user to view and edit the tracking lines generated and used by Analysis to collect data points for each sample. Individual Sample Files are stored for each of the samples analyzed and include the chromatogram, raw data, and annotations and information regarding the sample and sequence run. Generally, the products of a sequencing reaction are easily resolved and the Analysis software interprets the correct nucleotide sequence. Ambiguous base calls tend to occur near the end of the sequence and may be either edited or deleted by the user before exporting the data for further comparisons or alignments. Occasionally the tracking lines within the gel image may need to be adjusted or moved. The sample data are then reextracted from the Gel File and analyzed again. This review explains the general operation of Analysis in terms of viewing and editing a chromatogram, retracking the lanes of a Gel File, and analyzing the final sample data. The three versions 1.2.1, 2.1.2, and 3.3 are discussed.     

Specific-primer-directed DNA sequencing

A simple and rapid strategy for DNA sequence analysis based on the Sanger chain-termination method is described. This procedure utilizes full-sized inserts of 1 to 4 kb of DNA cloned into M13 bacteriophage vectors. After the sequence of the first 600-650 bp of the insert DNA has been determined with the commercially available universal vector primer, a specific oligonucleotide is synthesized utilizing the sequence data obtained from the 3' end of the sequence and used as a primer to extend the sequence analysis for another 600-650 nucleotides. Additional primers are synthesized in a similar manner until the nucleotide sequence of the entire insert DNA has been determined. General guidelines for the selection of oligonucleotide length and composition and the use of unpurified primers are discussed. The use of the specific-primer-directed approach to dideoxynucleotide sequence analysis, in association with highly purified single-stranded template DNA, reduces considerably the time required for the analysis of large segments of DNA.     

Mass-spectrometry DNA sequencing

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been explored widely for DNA sequencing. Compared to gel electrophoresis based sequencing systems, mass spectrometry produces very high resolution of sequencing fragments, rapid separation on microsecond time scales, and completely eliminates compressions associated with gel-based systems. While most of the research efforts have focused on using mass spectrometers to analyze the DNA products from Sanger sequencing or enzymatic digestion reactions, the read lengths attainable are currently insufficient for large-scale de novo sequencing. The advantage of mass-spectrometry sequencing is that one can unambiguously identify frameshift mutations and heterozygous mutations making it an ideal choice for resequencing projects. In these applications, DNA sequencing fragments that are the same length but with different base compositions are generated, which are challenging to consistently distinguish in gel-based sequencing systems. In contrast, MALDI-TOF MS produces mass spectra of these DNA sequencing fragments with nearly digital resolution, allowing accurate determination of the mixed bases. For these reasons mass spectrometry based sequencing has mainly been focused on the detection of frameshift mutations and single nucleotide polymorphisms (SNPs). More recently, assays have been developed to indirectly sequence DNA by first converting it into RNA. These assays take advantage of the increased resolution and detection ability of MALDI-TOF MS for RNA.     

Comparison of the experimental methods in haplotype sequencing via next generation sequencing

Although the diploid nature has been observed for over 50 years, phasing the diploid is still a laborious task. The speed and throughput of next generation sequencing have largely increased in the past decades. However, the short read-length remains one of the biggest challenges of haplotype analysis. For instance, reads as short as 150 bp span no more than one variant in most cases. Numerous experimental technologies have been developed to overcome this challenge. Distance, complexity and accuracy of the linkages obtained are the main factors to evaluate the efficiency of whole genome haplotyping methods. Here, we review these experimental technologies, evaluating their efficiency in linkages obtaining and system complexity. The technologies are organized into four categories based on its strategy: (i) chromosomes separation, (ii) dilution pools, (iii) crosslinking and proximity ligation, (ix) long-read technologies. Within each category, several subsections are listed to classify each technology. Innovative experimental strategies are expected to have high-quality performance, low cost and be labor-saving, which will be largely desired in the future.