TRANSCRIPTOME (RNA-SEQ) & SMALL RNA ANALYSIS
The complete sequencing of transcriptomes has become an important tool in the analysis of gene expression, alternative splice sites, allele specific expression and the discovery and analysis of rare or novel transcripts. Unlike the use of microarrays – which analyze known transcripts – the entire RNA population contained within the sample is sequenced by NGS. This approach yields an unprecedented amount of detail of the organization and structure of a transcriptome.
De novo transcriptome sequencing
De novo transcriptome sequencing allows the study of gene expression in any organism without prior knowledge of its genome sequence. RNA-Seq allows for a profiling of the whole population of RNA in any species and it enables mapping and digital quantification of whole transcripts. RNA-Seq also provides a rich source of sequence data for assessing alternative splice events.
De novo analysis requires higher levels of coverage in effort to provide a complete representation in unknown or uncharacterized samples. See Table 1 in Important Considerations for coverage-related issues.
As with whole genome sequencing, transcriptome analysis can be simplified when a reference transcriptome or genome has been made available. Levels of coverage may be reduced (see Table 1 in Important Considerations) and the reads can be mapped to the reference for SNP and indel analysis. Additionally, an accurate evaluation of transcript numbers can also be rendered.
Small RNA Discovery & Analysis
NGS technology has facilitated discovery and analysis of small, non-coding RNAs. Small RNA sequencing allows for the detection of all known and novel sequences, including the length and sequence variations, or isomirs, seen in microRNAs.
At ACGT, we routinely use the TruSeq Small RNA Sample Prep Kits from Illumina®, which provides an effective solution for generating small RNA libraries directly from total RNA. These kits enable multiplexed sequencing with the introduction of 48 unique indexes, allowing high levels of sample multiplexing. Kits from other providers may be used at the customer’s request.