Application of risk score analysis to low-coverage whole genome sequencing data for the noninvasive detection of trisomy 21, trisomy 18, and trisomy 13.

OBJECTIVES: Clinical performance of a low coverage, low cost, massively parallel sequencing (MPS)-based assay to stratify risk of trisomy 21, 18, and 13 pregnancies was determined. METHODS: The study included 1100 samples with birth outcome or karyotype results, comprising low-risk patients (84.2%) negative for risk indications from maternal age, serum screening, ultrasound, or family history, and high-risk patients (15.8%) with at least one of the aforementioned indications. Cell free DNA (cfDNA) was extracted from maternal plasma. Library preparation incorporated 96 index barcodes to enable sequencing on a HiSeq 2000 or 2500. Risk scores were calculated using chromosomal representation, fetal fraction, and maternal age at the estimated date of delivery. A risk score greater than or equal to 1 in 100 was used to stratify samples as high risk for trisomy 21, trisomy 18, or trisomy 13. RESULTS: Sensitivity and specificity were calculated based on risk group stratification. Trisomy 21, trisomy 18, and trisomy 13 were detected with greater than 99% sensitivity and 99.9% specificity. Fetal sex classification accuracy was 99.3%. CONCLUSIONS: We conclude that simplified MPS can be used to stratify the risk of pregnancies for trisomy 21, trisomy 18, and trisomy 13 and accurately determine fetal sex. © 2015 John Wiley & Sons, Ltd.

Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.

We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

In vitro cloning of complex mixtures of DNA on microbeads: physical separation of differentially expressed cDNAs.

We describe a method for cloning nucleic acid molecules onto the surfaces of 5-micrometer microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique tagging of the molecules is achieved by sampling a small fraction (1%) of a very large repertoire of tag sequences. The resulting library is hybridized to microbeads that each carry approximately 10(6) strands complementary to one of the tags. About 10(5) copies of each molecule are collected on each microbead. Because such clones are segregated on microbeads, they can be operated on simultaneously and then assayed separately. To demonstrate the utility of this approach, we show how to label and extract microbeads bearing clones differentially expressed between two libraries by using a fluorescence-activated cell sorter (FACS). Because no prior information about the cloned molecules is required, this process is obviously useful where sequence databases are incomplete or nonexistent. More importantly, the process also permits the isolation of clones that are expressed only in given tissues or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue- or disease-directed, low-density planar microarrays.

The NGF complex from the African rat Mastomys natalensis.

The 7S NGF complex from the male mouse submaxillary gland consists of the alpha, gamma, and beta subunits in the ratio alpha 2 gamma 2 beta. The beta (NGF) subunit contains all the known biolocial activity of 7S NGF. The alpha and gamma subunits are both members of glandular kallikrein gene family, yet only gamma subunit has protease activity. The gamma subunit plays a role in the processing of preproNGF to its mature form, while the role of the alpha subunit is not yet understood. Despite the fact that 7S NGF has been extensively characterized, no other NGF complex has been characterized, nor have the alpha or gamma subunits been observed in tissues which express NGF. We have therefore purified and characterized the NGF complex from the submaxillary glands of the rat Mastomys natalensis in order to more fully understand the roles of the alpha and gamma subunits. The NGF complex from M. natalensis contains subunits similar to those found in mouse 7S NGF. Although similar, there are significant differences between mouse and M. natalensis NGF complexes, especially in the degree of post-translational modification of the gamma and NGF subunits, the expression of esterase activity and the ease with which the complexes dissociate. Evidence is presented that suggests that the NGF complex from M. natalensis may consist of subunits in the ratio alpha 2 gamma beta. The amino acid sequence of the M. natalensis NGF suggests some, but not all, ways in which these differences arise.

Ice growth in supercooled solutions of antifreeze glycoprotein.

Inhibition of ice growth in supercooled solution by certain proteins is vital to the survival of many living organisms. Some fish, native to both subzero northern and southern waters, have special proteins or glycoproteins in their blood serum that inhibit ice formation. Whereas these proteins have only a very small effect on the melting temperature of ice, the temperature of these fish can fall to nearly 1 K below the melting point before ice crystals grow. This phenomenon is called freezing hysteresis, in contrast to the normal colligative effect of solutes that depresses the equilibrium temperature, around which small changes lead to crystal growth or melting depending on sign. Some insects also exhibit a serum freezing hysteresis. We report the effects of different degrees of supercooling on the habit and rates of growth of ice crystals from solutions of these antifreeze glycoproteins (AFGPs). We find that the crystallization rate is up to five times greater than that in pure water.

Purification and primary sequences of the major arginine-containing antifreeze glycopeptides from the fish Eleginus gracilis.

An Arg-containing antifreeze glycoprotein from the polar fish Eleginus gracilis was isolated, and the major components were purified to homogeneity. The general protocol for purification was chromatography of serum on DEAE-cellulose, followed by chromatography on a cation exchanger. DEAE-cellulose chromatography resulted in two fractions, A and B. Fraction A contained most of the antifreeze glycoprotein found in E. gracilis (approximately 80% by weight) and consisted of 13 distinct components. Unlike antifreeze glycoproteins from other previously studied polar fish, Fraction A contained both low and high molecular weight antifreeze glycoprotein components. The two major components of Fraction A were sequenced and compared with the sequence of antifreeze glycoproteins 7 and 8 from both Boreogadus saida and Pagothenia borchgrevinki. The antifreeze glycoproteins from E. gracilis were shown to have a similar composition to those previously studied, except for an additional Ala-Arg dipeptide at the carbon terminal in the major components of Fraction A and the position of Pro in the low molecular weight components. The activity of E. gracilis antifreeze glycoproteins is the subject of a companion article (Burcham, T. S., Osuga, D. T., Yeh, Y., and Feeney, R. E. (1986) J. Biol. Chem. 261, 6390-6397).

A kinetic description of antifreeze glycoprotein activity.

The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.

Antifreeze glycoproteins from polar fish blood.

Existing experimental evidence strongly suggests that the mechanism of activity of the Antarctic AFGP molecules is the inhibition of ice growth by competitive adsorption onto the growth sites of ice. The data further suggest the blocking of the formation of large critical nuclei for ice growth. Experiments showing that the longer polymers (AFGP 1-5) have different growth-prevention properties with different types of ice than the shorter polymers (AFGP 6-8) provide additional evidence that crystal size and habits are linked to function. Four main observations have been used in AFGP studies: (a) The ice crystal habit (size) affects the activity, (b) AFGP is on the surface of ice crystals, as shown by surface second harmonic generation (SSHG), (c) the presence of AFGP lowers the surface energy at the ice-solution interface, and (d) kinetic calculations of the inhibition of ice-crystal growth fit adsorption isotherms. In particular, AFGP 4 fits a Langmuirian adsorption curve. On the detailed mechanistic side, there is a need to quantify the competitive rates of water-molecule attachment to ice as well as the rates of adsorption-desorption of AFGP on the ice surface. Toward this end, experiments are currently being conducted to examine the differential growth rates of ice crystals freely growing into a solution of AFGP. Direct observation of the adsorption and desorption of AFGP on the ice surface is possible in principle using the SSHG technique. A nonperturbative probe method that can differentiate adsorbed AFGP from the solution phase molecules is needed. Finally, a mechanistic description of molecular function is never complete unless the detailed molecular binding to the surface is elucidated. This task has not been accomplished yet, and thus there is still a question as to which molecular group is actually adsorbed onto the surface and for how long. Theoretically, as we have seen, the departure from Langmuirian adsorption is pronounced for small AFGP molecules under certain conditions. More refinements of the ideas of intermolecular or intramolecular cooperative interaction of AFGP on the ice surface are needed.

Determination of tryptophan as the reduced derivative by acid hydrolysis and chromatography.

A new procedure for the analyses of tryptophan and the total amino acid composition of proteins was based on the observations that pyridine borane reduces tryptophan in trifluoroacetic acid, while other amino acids remain intact [M. Kurata, Y. Kikugawa, T. Kuwae, I. Koyama, and T. Takagi (1980) Chem. Pharm. Bull. 28, 2274-2275; W.S.D. Wong, D.T. Osuga, and R.E. Feeney (1984) Anal. Biochem. 139, 58-67]. Concentrated HCl was used instead of trifluoroacetic acid for analytical purposes. The products were stable to hydrolysis in 6 N HCl, and the reduction did not interfere with hydrolysis and subsequent analyses. Quantitative recovery was achieved with most proteins when they were subjected to acid reduction in ice-cooled concentrated HCl with two incremental additions of pyridine borane. The reaction was terminated after 10 min by dilution with an equal volume of H2O, vacuum sealing, and hydrolyzing at 110 degrees C for 22 h. The yields of the expected values for cytochrome c, catalase, bovine serum albumin, subtilisin BPN', trypsin, chymotrypsin, beta-lactoglobulin, lysozyme, and pepsin were obtained. Ovotransferrin and ovalbumin, however, yielded values for tryptophan lower than literature values. With two different ion-exchange methods, the recoveries of all other amino acids were comparable to those obtained by acid hydrolysis with 6 N HCl. Since the same hydrolysate can be analyzed for both tryptophan and all the other amino acids, the procedure is a more convenient method than those requiring separate determinations. Initial results indicate that the method may be applied to high-performance liquid chromatographic procedures with adaptations of the protocols if necessary.

Analysis of antifreeze glycoproteins in fish serum.

A procedure utilizing high-pressure size-exclusion chromatography that permits rapid screening for both the types of components present in and the quantity of antifreeze glycoprotein in fish serum or solution is described. The applicability of the method is demonstrated by a comparative study of five different fish species, four of which contain the antifreeze glycoprotein and one which does not contain this protein. The antifreeze glycoprotein compositions of two fish of the same species, collected at different locations or under different environmental conditions, are also compared. A linear molecular-weight versus elution-volume function is established for both standard native proteins and the antifreeze glycoproteins, but these two lines do not coincide. The differences in tertiary structure between the antifreeze glycoproteins and normal proteins are presented as an explanation for the nonequivalence of calibration lines.