Assessing a single targeted next generation sequencing for human leukocyte antigen typing protocol for interoperability,as performed by users with variable experience

Background

A simplified protocol for HLA-typing -by NGS, developed for use with the Illumina MiSeq, was performed by technologists with varying NGS experience to assess accuracy and reproducibility.

High prevalence of Brachyspira spp. in layers kept in alternative husbandry systems associated with frequent species variations from end of rearing to slaughter

A longitudinal survey was conducted to investigate the presence of Brachyspira species in layer flocks. A total of 66 layer flocks kept in alternative husbandry systems were sampled at three time points: end of rearing, at peak of lay and at end of lay. Content from caecal samples of freshly killed birds was cultured at each sampling time point and processed for further investigations. Gross pathological lesions in caeca were recorded during post mortem investigation. Spirochaetes were isolated from 50 flocks: three flocks were positive at all three sampling points, 25 flocks at two and 22 flocks at one sampling point, respectively. The presence of Brachyspira spp. could not be related to specific gross pathological caecal lesions or antibiotic treatments. The number of positive flocks increased with the age of birds. Furthermore, organic flocks were more often positive than flocks from barn systems. In total 80 spirochaetal cultures were obtained. B. intermedia (43.8%) was the most common species, followed by B. pulli (13.8%) and B. pilosicoli (12.5%). Brachyspira murdochii and B. innocens were found in 5.0% and 2.5%, respectively, whereas 11.3% of Brachyspira isolates could not be identified to species level. In 11.3% of the samples mixed infections were detected. Finally, the longitudinal survey revealed for the first time a possible shift in the Brachyspira species in a substantial number (32%) of layer flocks during their lifetime.     

组学大数据环境下的基因变异信息并行处理与分析

随着第二代测序技术的发展与应用,其产生的测序数据也呈现快速的增长趋势,如何有效、快速、稳定地对海量测序数据进行分析成为生物研究领域迫切的需求。目前许多传统的测序数据分析软件仅支持单一功能,并不具备完整的数据分析能力,应对海量的测序数据时其处理能力也显著不足。为了应对上述问题,本文设计了一款基于Hadoop框架的测序数据分析软件,整合了现今生物研究领域内常用的多款序列分析软件,从而实现了对测序序列数据的自动化分析。该软件输入原始的测序数据后,经过碱基质量控制、序列比对、SNP位点信息提取、突变基因信息生成等几个过程,最终输出详细的突变基因信息报告。该软件实现了自动化的数据分析,提高了数据分析的效率,极大减轻了数据分析人员的工作量。     

An introduction to microbiome analysis for human biology applications

Research examining the gut microbiota is currently exploding, and results are providing new perspectives on human biology. Factors such as host diet and physiology influence the composition and function of the gut microbiota, which in turn affects human nutrition, health, and behavior via interactions with metabolism, the immune system, and the brain. These findings represent an exciting new twist on familiar topics, and as a result, gut microbiome research is likely to provide insight into unresolved biological mechanisms driving human health. However, much remains to be learned about the broader ecological and evolutionary contexts within which gut microbes and humans are affecting each other. Here, I outline the procedures for generating data describing the gut microbiota with the goal of facilitating the wider integration of microbiome analyses into studies of human biology. I describe the steps involved in sample collection, DNA extraction, PCR amplification, high-throughput sequencing, and bioinformatics. While this review serves only as an introduction to these topics, it provides sufficient resources for researchers interested in launching new microbiome initiatives. As knowledge of these methods spreads, microbiome analysis should become a standard tool in the arsenal of human biology research.     

唐氏综合征患儿线粒体基因组突变的分析

目的 研究唐氏综合征中线粒体DNA突变情况.方法 采用高通量测序和焦磷酸测序检测7个唐氏综合征(Down's syndrome,DS)家系中的患儿和母亲的线粒体基因组序列,分析线粒体基因组序列的变化情况.结果 ①DS患儿中检测到36个与其母亲中不同的线粒体DNA突变,其中14个位点是首次在唐氏综合征样本中发现;②36个线粒体DNA突变主要发生于D-Loop区和线粒体复合物Ⅰ中;③ 线粒体基因组13个编码基因中,有11个基因检测到线粒体DNA的突变;④ 焦磷酸测序对线粒体基因组杂合突变频率的检测结果和高通量测序结果吻合.结论 DS患儿中广泛存在线粒体DNA的突变,这些突变可能与唐氏综合征的线粒体功能异常相关.     

Uncovering the genomic heterogeneity of multifocal breast cancer

Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non‐silent coding mutations in 360 protein‐coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as ‘oncogenic’ in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole‐genome rearrangement screen was further conducted in 8/36 patients. Twenty‐four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three‐quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter‐lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome‐wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter‐lesion heterogeneity in one‐third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers. © 2015 The Authors. Pathological Society of Great Britain and Ireland.     

Whole‐exome DNA sequence analysis of Brca2‐ and Trp53‐deficient mouse mammary gland tumours

Germline mutations in the tumour suppressor BRCA2 predispose to breast, ovarian and a number of other human cancers. Brca2‐deficient mouse models are used for preclinical studies but the pattern of genomic alterations in these tumours has not yet been described in detail. We have performed whole‐exome DNA sequencing analysis of mouse mammary tumours from Blg–Cre Brca2^f/f Trp53^f/f animals, a model of BRCA2‐deficient human cancer. We also used the sequencing data to estimate DNA copy number alterations in these tumours and identified a recurrent copy number gain in Met, which has been found amplified in other mouse mammary cancer models. Through a comparative genomic analysis, we identified several mouse Blg–Cre Brca2^f/f Trp53^f/f mammary tumour somatic mutations in genes that are also mutated in human cancer, but few of these genes have been found frequently mutated in human breast cancer. A more detailed analysis of these somatic mutations revealed a set of genes that are mutated in human BRCA2 mutant breast and ovarian tumours and that are also mutated in mouse Brca2‐null, Trp53‐null mammary tumours. Finally, a DNA deletion surrounded by microhomology signature found in human BRCA1/2‐deficient cancers was not common in the genome of these mouse tumours. Although a useful model, there are some differences in the genomic landscape of tumours arising in Blg–Cre Brca2^f/f Trp53^f/f mice compared to human BRCA‐mutated breast cancers. Therefore, this needs to be taken into account in the use of this model. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.