Next-Generation Sequencing — An Overview of the History, Tools, and “Omic” Applications

Next-generation sequencing (NGS) technologies using DNA, RNA, or methylation sequencing have impacted enormously on the life sciences. NGS is the choice for large-scale genomic and transcriptomic sequencing because of the high-throughput production and outputs of sequencing data in the gigabase range per instrument run and the lower cost compared to the traditional Sanger first-generation sequencing method. The vast amounts of data generated by NGS have broadened our understanding of structural and functional genomics through the concepts of “omics” ranging from basic genomics to integrated systeomics, providing new insight into the workings and meaning of genetic conservation and diversity of living things. NGS today is more than ever about how different organisms use genetic information and molecular biology to survive and reproduce with and without mutations, disease, and diversity within their population networks and changing environments. In this chapter, the advances, applications, and challenges of NGS are reviewed starting with a history of first-generation sequencing followed by the major NGS platforms, the bioinformatics issues confronting NGS data storage and analysis, and the impacts made in the fields of genetics, biology, agriculture, and medicine in the brave, new world of ”omics.

The teaching portfolio project: An evaluative case study of a portfolio-based approach to the development of university teaching

This study on the use of teaching portfolios arose from a number of converging trends and policy initiatives within the higher education sector that led to demands for the improvement of, and a more reflective scholarly approach to, university teaching. In Australia, and overseas, institutions have responded to these demands by implementing teaching development and evaluation programs for academic staff that arc based on the use of portfolios. A teaching portfolio is essentially a documentary record of selected aspects of a teacher\u27s work across a range of instructional settings. According to some proponents, portfolios can capture the complexity of university teaching in a manner that is both discipline-based and context-specific and thus offer advantages over traditional approaches to teaching appraisal and improvement. However, as portfolios are a relatively recent phenomenon in higher education, their increasing use for both summative and formative evaluation of higher education staff raises a number of questions and concerns. Against this background, the present study explored the role of teaching portfolios in the professional development of academic staff and the appraisal and improvement of teaching quality. It did so through an evaluative case study of a Teaching Portfolio Project (TPP) that involved the planning, implementation and evaluation of a Staff Development Program (SDP) for academic staff in the School of Nursing at Curtin University of Technology. Stufflebeam\u27s CIPP evaluation model, comprising discrete context, input, process and product evaluations, provided the framework for informing the design of the SDP and for a comprehensive investigation of the issues surrounding the use of teaching portfolios in a university setting. The study has shed substantial light on the usefulness of portfolio-based approaches to teaching development. The findings show that with careful planning and appropriate resources a portfolio-based staff development program can be successfully implemented in a university department and point the way to introducing similar initiatives across the university. They also provide insight into how portfolio preparation may be integrated with existing institutional practices for teaching improvement and appraisal, and how portfolios can be adapted to document teaching across a range of instructional settings. Taken together, the findings of the present study demonstrate that the process of portfolio preparation provides a useful approach to the appraisal and improvement of university teaching and can be a powerful and engaging strategy for academic staff development. The findings further demonstrate that the preparation of a portfolio can facilitate reflective teaching practice and improvement, and that group-based approaches can promote a collegial discourse for teaching development. Whilst the findings of the TPP show that portfolio use in higher education appears to fulfil its early promise, they also highlight areas that will require further investigation

MHC Genotyping in Human and Nonhuman Species by PCRbased Next-Generation Sequencing

The major histocompatibility complex (MHC) is a highly polymorphic genomic region that encodes the transplantation and immune regulatory molecules. It receives special attention for genetic investigation because of its important role in the regulation of innate and adaptive immune responses and its strong association with numerous infectious and/or autoimmune diseases. Recently, genotyping of the polymorphisms of MHC genes using targeted next-generation sequencing (NGS) technologies was developed for humans and some nonhuman species. Most species have numerous highly homologous MHC loci so the NGS technologies are likely to replace traditional genotyping methods in the near future for the investigation of human and animal MHC genes in evolutionary biology, ecology, population genetics, and disease and transplantation studies. In this chapter, we provide a short review of the use of targeted NGS for MHC genotyping in humans and nonhuman species, particularly for the class I and class II regions of the Crab-eating Macaque MHC (Mafa)

Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism

ArticleNATURE PROTOCOLS. 2(11): 2857-2864 (2007)journal articl

Major histocompatibility complex (Mhc) class Ib gene duplications, organization and expression patterns in mouse strain C57BL/6

<p>Abstract</p> <p>Background</p> <p>The mouse has more than 30 <it>Major histocompatibility complex </it>(<it>Mhc</it>) class Ib genes, most of which exist in the <it>H2 </it>region of chromosome 17 in distinct gene clusters. Although recent progress in <it>Mhc </it>research has revealed the unique roles of several <it>Mhc </it>class Ib genes in the immune and non-immune systems, the functions of many class Ib genes have still to be elucidated. To better understand the roles of class Ib molecules, we have characterized their gene duplication, organization and expression patterns within the <it>H2 </it>region of the mouse strain C57BL/6.</p> <p>Results</p> <p>The genomic organization of the <it>H2-Q</it>, -<it>T </it>and -<it>M </it>regions was analyzed and 21 transcribed <it>Mhc </it>class Ib genes were identified within these regions. Dot-plot and phylogenetic analyses implied that the genes were generated by monogenic and/or multigenic duplicated events. To investigate the adult tissue, embryonic and placental expressions of these genes, we performed RT-PCR gene expression profiling using gene-specific primers. Both tissue-wide and tissue-specific gene expression patterns were obtained that suggest that the variations in the gene expression may depend on the genomic location of the duplicated genes as well as locus specific mechanisms. The genes located in the <it>H2-T </it>region at the centromeric end of the cluster were expressed more widely than those at the telomeric end, which showed tissue-restricted expression in spite of nucleotide sequence similarities among gene paralogs.</p> <p>Conclusion</p> <p>Duplicated <it>Mhc </it>class Ib genes located in the <it>H2-Q</it>, -<it>T </it>and -<it>M </it>regions are differentially expressed in a variety of developing and adult tissues. Our findings form the basis for further functional validation studies of the <it>Mhc </it>class Ib gene expression profiles in specific tissues, such as the brain. The duplicated gene expression results in combination with the genome analysis suggest the possibility of long-range regulation of <it>H2-T </it>gene expression and/or important, but as yet unidentified nucleotide changes in the promoter or enhancer regions of the genes. Since the <it>Mhc </it>genomic region has diversified among mouse strains, it should be a useful model region for comparative analyses of the relationships between duplicated gene organization, evolution and the regulation of expression patterns.</p

Compound Evolutionary History of the Rhesus Macaque Mhc Class I B Region Revealed by Microsatellite Analysis and Localization of Retroviral Sequences

In humans, the single polymorphic B locus of the major histocompatibility complex is linked to the microsatellite MIB. In rhesus macaques, however, haplotypes are characterized by the presence of unique combinations of multiple B genes, which may display different levels of polymorphism. The aim of the study was to shed light on the evolutionary history of this highly complex region. First, the robustness of the microsatellite MIB-linked to almost half of the B genes in rhesus macaques (Mamu-B)–for accurate B haplotyping was studied. Based on the physical map of an established haplotype comprising 7 MIB loci, each located next to a certain Mamu-B gene, two MIB loci, MIB1 and MIB6, were investigated in a panel of MHC homozygous monkeys. MIB1 revealed a complex genotyping pattern, whereas MIB6 analysis resulted in the detection of one or no amplicon. Both patterns are specific for a given B haplotype, show Mendelian segregation, and even allow a more precise haplotype definition than do traditional typing methods. Second, a search was performed for retroelements that may have played a role in duplication processes as observed in the macaque B region. This resulted in the description of two types of duplicons. One basic unit comprises an expressed Mamu-B gene, adjacent to an HERV16 copy closely linked to MIB. The second type of duplicon comprises a Mamu-B (pseudo)gene, linked to a truncated HERV16 structure lacking its MIB segment. Such truncation seems to coincide with the loss of B gene transcription. Subsequent to the duplication processes, recombination between MIB and Mamu-B loci appears to have occurred, resulting in a hyperplastic B region. Thus, analysis of MIB in addition to B loci allows deciphering of the compound evolutionary history of the class I B region in Old World monkeys

Nucleotide alterations in the HLA-C class I gene can cause aberrant splicing and marked changes in RNA levels in a polymorphic context-dependent manner

Polymorphisms of HLA genes, which play a crucial role in presenting peptides with diverse sequences in their peptide-binding pockets, are also thought to affect HLA gene expression, as many studies have reported associations between HLA gene polymorphisms and their expression levels. In this study, we devised an ectopic expression assay for the HLA class I genes in the context of the entire gene, and used the assay to show that the HLA-C*03:03:01 and C*04:01:01 polymorphic differences observed in association studies indeed cause different levels of RNA expression. Subsequently, we investigated the C*03:23N null allele, which was previously noted for its reduced expression, attributed to an alternate exon 3 3’ splice site generated by G/A polymorphism at position 781 within the exon 3. We conducted a thorough analysis of the splicing patterns of C*03:23N, and revealed multiple aberrant splicing, including the exon 3 alternative splicing, which overshadowed its canonical counterpart. After confirming a significant reduction in RNA levels caused by the G781A alteration in our ectopic assay, we probed the function of the G-rich sequence preceding the canonical exon 3 3’ splice site. Substituting the G-rich sequence with a typical pyrimidine-rich 3’ splice site sequence on C*03:23N resulted in a marked elevation in RNA levels, likely due to the enhanced preference for the canonical exon 3 3’ splice site over the alternate site. However, the same substitution led to a reduction in RNA levels for C*03:03:01. These findings suggested the dual roles of the G-rich sequence in RNA expression, and furthermore, underscore the importance of studying polymorphism effects within the framework of the entire gene, extending beyond conventional mini-gene reporter assays