Capturing needles in haystacks: a comparison of B-cell receptor sequencing methods.

BACKGROUND: Deep-sequencing methods are rapidly developing in the field of B-cell receptor (BCR) and T-cell receptor (TCR) diversity. These promise to revolutionise our understanding of adaptive immune dynamics, identify novel antibodies, and allow monitoring of minimal residual disease. However, different methods for BCR and TCR enrichment and amplification have been proposed. Here we perform the first systematic comparison between different methods of enrichment, amplification and sequencing for generating BCR and TCR repertoires using large sample numbers. RESULTS: Resampling from the same RNA or cDNA pool results in highly correlated and reproducible repertoires, but resampling low frequency clones leads to stochastic variance. Repertoires generated by different sequencing methods (454 Roche and Illumina MiSeq) and amplification methods (multiplex PCR, 5' Rapid amplification of cDNA ends (5'RACE), and RNA-capture) are highly correlated, and resulting IgHV gene frequencies between the different methods were not significantly different. Read length has an impact on captured repertoire structure, and ultimately full-length BCR sequences are most informative for repertoire analysis as diversity outside of the CDR is very useful for phylogenetic analysis. Additionally, we show RNA-based BCR repertoires are more informative than using DNA. CONCLUSIONS: Repertoires generated by different sequencing and amplification methods are consistent, but we show that read lengths, depths and error profiles should be considered in experimental design, and multiple sampling approaches could be employed to minimise stochastic sampling variation. This detailed investigation of immune repertoire sequencing methods is essential for informing basic and clinical research.

The role of high-throughput technologies in clinical cancer genomics.

Cancer is a genetic disease driven by both heritable and somatic alterations in DNA, which underpin not only oncogenesis but also progression and eventual metastasis. The major impetus for elucidating the nature and function of somatic mutations in cancer genomes is the potential for the development of effective targeted anticancer therapies. Over the last decade, high-throughput technologies have allowed us unprecedented access to a host of cancer genomes, leading to an influx of new information about their pathobiology. The challenge now is to integrate such emerging information into clinical practice to achieve tangible benefits for cancer patients. This review examines the roles array-based comparative genomic hybridization and next-generation sequencing are playing in furthering our understanding of both hematological and solid-organ tumors. Furthermore, the authors discuss the current challenges in translating the role of these technologies from bench to bedside.

Cholangiocarcinoma presenting as hemobilia and recurrent iron-deficiency anemia: a case report.

INTRODUCTION: Iron-deficiency anemia is a relatively common presenting feature of several gastrointestinal malignancies. However, cholangiocarcinoma has rarely been reported as an underlying cause. The association of cholangiocarcinoma with the rare clinical finding of hemobilia is also highly unusual. To our knowledge, this is the first case report of cholangiocarcinoma presenting with acute hemobilia and chronic iron-deficiency anemia. CASE PRESENTATION: We report the case of a Caucasian, 84-year-old woman presenting with recurrent, severe iron-deficiency anemia who was eventually diagnosed with intra-hepatic cholangiocarcinoma, following an acute episode of hemobilia. A right hepatectomy was subsequently performed with curative intent, and our patient has now fully recovered. CONCLUSION: This is a rare example of hemobilia and chronic iron-deficiency anemia in association with cholangiocarcinoma. We suggest that a diagnosis of cholangiocarcinoma should be considered in patients who present with iron-deficiency anemia of unknown cause, particularly in the presence of abnormal liver function.