Michael W. Hunkapiller

Resolving the complexity of the human genome using single-molecule sequencing

The human genome is arguably the most complete mammalian reference assembly, yet more than 160 euchromatic gaps remain and aspects of its structural variation remain poorly understood ten years after its completion. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome—78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5xa0kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long short tandem repeats. Our results suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read sequencing technology.

Whole-genome shotgun assembly and comparison of human genome assemblies

We report a whole-genome shotgun assembly (called WGSA) of the human genome generated at Celera in 2001. The Celera-generated shotgun data set consisted of 27 million sequencing reads organized in pairs by virtue of end-sequencing 2-kbp, 10-kbp, and 50-kbp inserts from shotgun clone libraries. The quality-trimmed reads covered the genome 5.3 times, and the inserts from which pairs of reads were obtained covered the genome 39 times. With the nearly complete human DNA sequence [National Center for Biotechnology Information (NCBI) Build 34] now available, it is possible to directly assess the quality, accuracy, and completeness of WGSA and of the first reconstructions of the human genome reported in two landmark papers in February 2001 [Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A., et al. (2001) Science 291, 1304–1351; International Human Genome Sequencing Consortium (2001) Nature 409, 860–921]. The analysis of WGSA shows 97% order and orientation agreement with NCBI Build 34, where most of the 3% of sequence out of order is due to scaffold placement problems as opposed to assembly errors within the scaffolds themselves. In addition, WGSA fills some of the remaining gaps in NCBI Build 34. The early genome sequences all covered about the same amount of the genome, but they did so in different ways. The Celera results provide more order and orientation, and the consortium sequence provides better coverage of exact and nearly exact repeats.

Automated DNA sequencing and analysis of the human genome

The Human Genome Initiative is a complex, multifaceted, international effort to establish a massive data base of map and sequence information for humans and other organisms. The success of this initiative is dependent upon the development of new technologies for the analysis of genomes. In this paper, an overview of the Human Genome Initiative is presented, and the current status of efforts to automate large-scale DNA sequence analysis is reviewed.

Utility of the gas-phase sequencer for both liquid- and solid-phase degradation of proteins and peptides at low picomole levels

The utility of the commercially available gas-phase sequencer for complete analysis of peptide samples was investigated. Using the program supplied with the instrument, significant extractive loss of samples in Polybrene was observed, even at input levels up to 500 pmol. In order to reduce this loss, the sequencer program was modified by increasing the phenylisothiocyanate (PITC)-coupling steps from two to three and lengthening the duration of ethyl acetate (S2) delivery while reducing the delivery rate. These changes gave improved results with peptides, e.g., all eight residues of angiotensin II were identified at the 25-pmol level. In addition, background contamination was decreased and repetitive yields were increased. The instrument was also found to function well with samples coupled to solid supports; however, some of the methodologies that work adequately for covalent attachment of peptides to solid supports at the level 1-10 nmol were found to give unacceptable coupling/sequenceable yields at or below the 100-pmol level. The coupling methods tried were (1) reaction of homoserine lactone with aminopropyl (AP)-glass, (2) reaction of alpha- and epsilon-NH2 groups with p-phenylenediisothiocyanate (DITC)-glass, and (3) reaction of alpha-COOH groups with aminoaryl (AA)-glass via EDAC (1-ethyl-3,3-dimethylaminopropyl-carbodiimide). Of these, the first method gave combined yields of 42-94% while the latter two were only 9-35% efficient. The covalently bound samples provided sequence information even at the resulting low levels, e.g., 9/13 residues of dynorphin including Lys-13 at 11 pmol. In general, sequencer runs on solid-phase samples gave cleaner analyses and slightly higher repetitive yields (1-2%). Sequence information has also been obtained on peptides made by solid-phase synthesis prior to cleavage from the polystyrene support. With improved coupling efficiencies, solid-phase techniques would provide an alternative to immobilization of peptides in Polybrene films for low picomole level gas-phase sequencing.

Advances in DNA sequencing technology

Efforts to map and sequence the genomes of the human and other species have stimulated efforts to improve the technology required for these endeavors. During the last year, these efforts have produced substantial advances in DNA template preparation, sequencing chemistry, and gel analysis.

Applications of tandem microbore liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/electroblotting in microsequence analysis.

Protein isolation by microbore HPLC is compared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/electroblotting methods for several major proteins from rabbit muscle. Although single-mode HPLC or SDS-PAGE/electroblotting provides excellent speed and sensitivity for submicrogram-level protein purification, neither one alone has adequate resolution for separating such a complex protein mixture. Tandem procedures, utilizing two different modes of HPLC in separate steps or a combination of single HPLC separation and SDS-PAGE/electroblotting, offer the necessary versatility. One of the major concerns in this investigation was to evaluate electroblotting techniques for microsequencing. The Aebersold et al. procedure (R.H. Aebersold, D.B. Teplow, L.E. Hood, and S.B.H. Kent (1986) J. Biol. Chem. 261, 4229-4238) was substantially modified and improved; the details of this work will be published elsewhere. These changes significantly improve repetitive yields at the low microgram level without producing high backgrounds. At lower levels the recovery of sequenceable protein currently limits our ability to obtain useful results. Starting with 250-750 micrograms of rabbit muscle crude extract, several proteins (15-70 kDa) were isolated by tandem microbore LC and PAGE/electroblotting for amino-terminal sequence analysis. It appears that the combination of electroblotting and microbore LC represents a powerful approach for microsample preparation.

PTH Amino Acid Analysis

The chemical process, employed by automated protein/peptide sequencers is derived from the technique originated by Pehr Edman in the 1950s for the sequential degradation of peptide chains.1, 2. The first step in this degradation is selective coupling of a peptide’s amino-terminal amino acid with the Edman reagent, phenylisothiocyanate (PITC), a reaction catalyzed by an organic base delivered with the coupling reagent. The second step is cleavage of this derivatized amino acid from the remainder of the peptide, a reaction effected by treating the peptide with a strong organic acid. Each repeated coupling/cleavage cycle occurs at the newly-formed amino-terminal amino acid left by the previous cycle. Thus, repetitive cycles provide sequential separation of the amino acids which form the primary structure of the peptide.

Identification of a long form of cystatin from human saliva by rapid microbore HPLC mapping.

Microbore HPLC methodology permits rapid and sensitive mapping of human saliva proteins. Saliva is sampled and processed in less than one hour, greatly reducing the likelihood of artifactual protein degradation. As little as 50 microliters of saliva yields proteins in sufficient quantities and purity to obtain amino terminal sequences directly. By this route we have discovered a 14 kDa protein extremely homologous to Cystatin S, but amino-terminally extended by eight amino acids.

Automated Amino Acid Sequence Assignment: Development of a Fully Automated Protein Sequencer Using Edman Degradation

The chemical process employed by automated protein/peptide sequencers derives from the technique originated by Pehr Edman in the 1950s for the sequential degradation of peptide chains (1, 2). The first step in this degradation is selective coupling of a peptide’s amino-terminal amino acid with the Edman reagent, phenylisothiocyanate (PITC), a reaction catalyzed by an organic base delivered with the coupling reagent. The second step is cleavage of this derivatized amino acid from the remainder of the peptide, a reaction effected by treating the peptide with a strong organic acid. Each repeated coupling/cleavage cycle occurs at the newly-formed amino-terminal amino acid left by the previous cycle. Thus, repetitive cycles provide sequential separation of the amino acids which form the primary structure of the peptide.

Obtaining Primary Sequence Information from the Carboxy Terminal End of Polypeptides

The principles of end labeling have been firmly established in the DNA sequencing field (for example, Smith et. al., 1986). In fact, end labelling in conjunction with an appropriate separation technique is the cornerstone of this technology. Unfortunately, the intrinsically higher chemical diversity of proteins built from 20+ different amino acids (compared with the 4 bases in DNA) with a wide range of chemical reactivity has impeded the development of such techniques for protein chemists. Such procedures would be useful in both finding the C-terminal fragment for sequence analysis, and making alignments from partial cleavage maps. Selective isolation followed by amino terminal sequencing is an alternative to direct chemical sequencing from the carboxy terminal.