Reducing Biases in Small RNA Sequencing
Poster Feb 19, 2015
Adam Morris Ph.D., Dawn Obermoeller, Masoud Toloue Ph.D.
The past decade has seen an explosion of interest in cataloging the small RNA repertoires of animal and plant species, and in understanding the biological function of small RNAs. Distinguishing closely-related small RNAs is difficult using hybridization-based approaches, since imperfectly matched small RNAs may hybridize to primers or immobilized probes. These considerations have led to the realization that high throughput sequencing is the most practical method for large-scale small RNA studies that aim to identify and enumerate small RNAs in various species and tissues.
Unfortunately, NGS approaches for small RNA analysis are not without their own challenges. Several studies have now shown entire datasets, including those in miRBase to contain severe sequence bias, specifically, small RNA expression that is not accurately represented by sRNA-seq. Significant effort has gone into identifying the cause of this misrepresentation, and it is now generally accepted that bias in sRNA-seq libraries is primarily introduced during the ligation steps in library preparation. Specifically, RNA ligases show sequence-specific preferences toward flow cell adapters, resulting in preferential inclusion of some small RNAs in sRNA-seq libraries, at the expense of others. Simply using two different adapter sequences during ligation can result in up to 30-fold differential expression for some microRNAs. No single adapter sequence is able to efficiently ligate to all small RNAs, indicating that the target sequence, as well as adapter sequence, is a source of bias.
Our approach to overcoming ligation bias in sRNA-seq libraries involves using a pool of adapters having randomized sequences at the ligation site, where each adapter sequence is present in vast molar excess over any given small RNA in the sample. Experiments show that most of the bias in adapter ligation is due to the sequence of 2-4 adapter nucleotides adjacent to the target junction.
Using our randomized adapter strategy, small RNA libraries were prepared with both synthetic small RNAs and small RNA isolated from human brain and sequenced. Libraries utilizing randomized adapters demonstrated significantly more even coverage due to reductions in ligase bias. We will demonstrate why our new streamlined small RNA-seq protocol is critical for those needing to accurately assess small RNA abundance using high throughput sequencing.
Characterization of a Type 2 diabetes-associated islet-specific enhancer cluster in STARD10 by genome editing of EndoC-βH1 cellsPoster
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
Psychiatric Risk Gene Cacna1c and Early Life Stress: Potential Gene-Environment interactions?Poster
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.
Correlations among the Toddler Gut Microbiome and Health Status VariablesPoster
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