Improved Small RNA Library Preparation Workflow for Next-Generation Sequencing
Poster May 06, 2015
Sabrina Shore, Jordana Henderson, Anton McCaffrey, Gerald Zon, Richard Hogrefe
Next-generation sequencing (NGS) is used to analyze microRNA (miRNA), a class of small non-coding RNAs that are important therapeutic targets and diagnostic markers. Commercially available small RNA sequencing library preparation kits require large inputs (>100 ng) and a laborious gel purification step. Additionally, commercial kits are hindered by adapter dimer formation, where 5' and 3' adapters ligate without an intervening RNA insert. Adapter dimers preferentially amplify during PCR. This is exacerbated at low RNA inputs where adapter dimer formation can greatly diminish usable sequencing reads.
We describe an optimized workflow which suppresses adapter dimers, works with low RNA input and eliminates the need for a gel purification step. Our workflow introduces chemically modified adapters that efficiently form libraries while reducing adapter dimer formation. TriLink’s modified adapter workflow allows RNA inputs as low as 1 ng with less than 1% adapter dimer reads when gel purified. Furthermore, when our workflow is combined with bead-based size selection purification, automation becomes possible.
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
Early life stress (ELS) is highly associated with development of psychopathology
and mood disorders in adulthood. Genetic studies have identified variation in the gene calcium voltage-gated channel subunit alpha1C (CACNA1C) to increase risk for several psychiatric disorders. This poster assessed the expression of Cacna1c following prepubertal stress.
Establishment and succession of the human microbiome begins at birth and microbial composition adapts alongside human development and growth. Development of a healthy signature microbial community has significant effects on health.This study followed 18 very low birth weight (VLBW) infants, measured initially in the from Neonatal Intensive Care Unit (NICU), at the age of two to three years old.READ MORE