Variation in the major histocompatibility complex (MHC) is increasingly associated with disease susceptibility and resistance in avian species of agricultural importance. This variation includes sequence polymorphisms but also structural differences (gene rearrangement) and copy number variation (CNV). The MHC has now been described for multiple galliform species including the best defined assemblies of the chicken (Gallus gallus) and domestic turkey (Meleagris gallopavo). Using this sequence resource, this study applied high-throughput sequencing to investigate MHC variation in turkeys of North America (NA turkeys). An MHC-specific SureSelect (Agilent) capture array was developed, and libraries were created for 14 turkeys representing domestic (commercial bred), heritage breed, and wild turkeys. In addition, a representative of the Ocellated turkey (M. ocellata) and chicken (G. gallus) was included to test cross-species applicability of the capture array allowing for identification of new species-specific polymorphisms. Libraries were hybridized to ∼12 K cRNA baits and the resulting pools were sequenced. On average, 98% of processed reads mapped to the turkey whole genome sequence and 53% to the MHC target. In addition to the MHC, capture hybridization recovered sequences corresponding to other MHC regions. Sequence alignment and de novo assembly indicated the presence of several additional BG genes in the turkey with evidence for CNV. Variant detection identified an average of 2245 polymorphisms per individual for the NA turkeys, 3012 for the Ocellated turkey, and 462 variants in the chicken (RJF-256). This study provides an extensive sequence resource for examining MHC variation and its relation to health of this agriculturally important group of birds.

Targeted capture enrichment and sequencing identifies extensive nucleotide variation in the turkey MHC- B

Sudden cardiac death in people between the ages of 1–40 years is a devastating event and is frequently caused by several heritable cardiac disorders. These disorders include cardiac ion channelopathies, such as long QT syndrome, catecholaminergic polymorphic ventricular tachycardia and Brugada syndrome and cardiomyopathies, such as hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy. Through careful molecular genetic evaluation of DNA from sudden death victims, the causative gene mutation can be uncovered, and the rest of the family can be screened and preventative measures implemented in at-risk individuals. The current screening approach in most diagnostic laboratories uses Sanger-based sequencing; however, this method is time consuming and labour intensive. The development of massively parallel sequencing has made it possible to produce millions of sequence reads simultaneously and is potentially an ideal approach to screen for mutations in genes that are associated with sudden cardiac death. This approach offers mutation screening at reduced cost and turnaround time. Here, we will review the current commercially available enrichment kits, massively parallel sequencing (MPS) platforms, downstream data analysis and its application to sudden cardiac death in a diagnostic environment.

https://www.mdpi.com/2076-3271/2/2/98/pdf

Application of Massively Parallel Sequencing in the Clinical Diagnostic Testing of Inherited Cardiac Conditions

/pdf/multiple-molecular-mechanisms-contribute-towards-in-vitro-29vsrbm0xj.pdf

Multiple Molecular Mechanisms Contribute Towards In Vitro Resistance to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukaemia

Advancement in next generation sequencing offers the possibility of routine use of whole genome sequencing (WGS) for Mycobacterium bovis (M. bovis) genomes in clinical reference laboratories. To date, the M. bovis genome could only be sequenced if the mycobacteria were cultured from tissue. This requirement for culture has been due to the overwhelmingly large amount of host DNA present when DNA is prepared directly from a granuloma. To overcome this formidable hurdle, we evaluated the usefulness of an RNA-based targeted enrichment method to sequence M. bovis DNA directly from tissue samples without culture. Initial spiking experiments for method development were established by spiking DNA extracted from tissue samples with serially diluted M. bovis BCG DNA at the following concentration range: 0.1 ng/μl to 0.1 pg/μl (10–1 to 10–4). Library preparation, hybridization and enrichment was performed using SureSelect custom capture library RNA baits and the SureSelect XT HS2 target enrichment system for Illumina paired-end sequencing. The method validation was then assessed using direct WGS of M. bovis DNA extracted from tissue samples from naturally (n = 6) and experimentally (n = 6) infected animals with variable Ct values. Direct WGS of spiked DNA samples achieved 99.1% mean genome coverage (mean depth of coverage: 108×) and 98.8% mean genome coverage (mean depth of coverage: 26.4×) for tissue samples spiked with BCG DNA at 10–1 (mean Ct value: 20.3) and 10–2 (mean Ct value: 23.4), respectively. The M. bovis genome from the experimentally and naturally infected tissue samples was successfully sequenced with a mean genome coverage of 99.56% and depth of genome coverage ranging from 9.2× to 72.1×. The spoligoyping and M. bovis group assignment derived from sequencing DNA directly from the infected tissue samples matched that of the cultured isolates from the same sample. Our results show that direct sequencing of M. bovis DNA from tissue samples has the potential to provide accurate sequencing of M. bovis genomes significantly faster than WGS from cultures in research and diagnostic settings.

/pdf/whole-genome-sequencing-of-mycobacterium-bovis-directly-from-5zkn8o9l.pdf

Whole genome sequencing of Mycobacterium bovis directly from clinical tissue samples without culture

The dramatic increase in throughput of sequencing data from next generation sequencing platforms has enabled scientists to study the genome with unprecedented depth and accuracy. Nevertheless, routine genetic screens in large numbers of individuals continue to remain costprohibitive through these approaches. Agilent Technologies' SureSelect platform for targeted exome capture, combined with massively parallel sequencing, provides a more affordable method to gain novel insights into the genetic causes of inherited disorders. In addition, identification of both common and rare polymorphisms implicated in complex diseases like cancer is greatly facilitated by selectively sequencing the protein-coding regions of the genome. In collaboration with the Broad and Sanger Institutes, Agilent Technologies has continued to expand the number of SureSelect target enrichment catalog products in order to enable a more comprehensive view of the protein-coding regions in humans and model organisms. We discuss the SureSelectHuman All Exon v2 (44Mb) and SureSelectHuman All Exon 50Mb designs. We also introduce the SureSelectMouse All Exon target enrichment system, which improves the ability to study genetic variation between strains in greater detail, and significantly increases the efficiency of screening for causative mutations in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. We demonstrate high performance with respect to capture efficiency, uniformity, reproducibility of enrichment, and ability to detect SNPs, insertion/deletions, and CNVs across Illumina (Genome Analyzer IIx and HiSeq2000) and SOLiD platforms. We highlight the utility of the SureSelect All Exon product portfolio for a wide variety of applications primarily due to the high specificity and excellent cross-platform sequence coverage. SureSelect All Exon designs also provide a means for standardization, consistency of performance, and reliability across multiple laboratories.

The Agilent Technologies' SureSelect™ All Exon Product Portfolio: High Performance Target Enrichment System for Human and Mouse Exome Sequencing on Illumina and SOLiD Platforms

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The Agilent Technologies' SureSelect™ All Exon Product Portfolio: High Performance Target Enrichment System for Human and Mouse Exome Sequencing on Illumina and SOLiD Platforms