Next-Generation Sequencing RNA-Seq Library Construction.

This unit presents protocols for construction of next-generation sequencing (NGS) directional RNA sequencing libraries for the Illumina HiSeq and MiSeq from a wide variety of input RNA sources. The protocols are based on the New England Biolabs (NEB) small RNA library preparation set for Illumina, although similar kits exist from different vendors. The protocol preserves the orientation of the original RNA in the final sequencing library, enabling strand-specific analysis of the resulting data.

B cell exchange across the blood-brain barrier in multiple sclerosis.

In multiple sclerosis (MS) pathogenic B cells likely act on both sides of the blood-brain barrier (BBB). However, it is unclear whether antigen-experienced B cells are shared between the CNS and the peripheral blood (PB) compartments. We applied deep repertoire sequencing of IgG heavy chain variable region genes (IgG-VH) in paired cerebrospinal fluid and PB samples from patients with MS and other neurological diseases to identify related B cells that are common to both compartments.

Naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation.

A diverse antibody repertoire is essential for an effective adaptive immune response to novel molecular surfaces. Although past studies have observed common patterns of V-segment use, as well as variation in V-segment use between individuals, the relative contributions to variance from genetics, disease, age, and environment have remained unclear. Using high-throughput sequence analysis of monozygotic twins, we show that variation in naive V(H) and D(H) segment use is strongly determined by an individual's germ-line genetic background.

Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire.

Antibody repertoire diversity, potentially as high as 10(11) unique molecules in a single individual, confounds characterization by conventional sequence analyses. In this study, we present a general method for assessing human antibody sequence diversity displayed on phage using massively parallel pyrosequencing, a novel application of Kabat column-labeled profile Hidden Markov Models, and translated complementarity determining region (CDR) capture-recapture analysis. Pyrosequencing of domain amplicon and RCA PCR products generated 1.