Transcriptome (RNA-Seq) and Small RNA Analysis    

The use of a sequencing approach for transcriptome analysis 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 microarray approach which analyzes the known transcripts contained on the array, with the NGS approach the entire RNA population contained in the sample is sequenced. This provides an unprecedented view of the transcriptome.

De novo transcriptome sequencing

This approach allows the study of gene expression in ANY organism without any prior knowledge of gene sequence! RNA-Seq allows the profiling of the whole population of mRNA in any eukaryotic species and enables mapping and digital quantification of whole transcripts. It also provides a rich source of sequence data for assessing alternative splice events.

De novo analysis requires higher levels of coverage in order to ensure complete representation in unknown or uncharacterized samples. See Table 2 below and Important Considerations for coverage-related issues.

Transcriptome Re-sequencing

As with the whole genome sequencing, transcriptome analysis is simplified if a reference transcriptome is available. Reduced levels of coverage can be used, and the reads are mapped to the reference for SNP and InDel analysis. An accurate evaluation of transcript numbers can also be made.

Small RNA Discovery & Analysis

Discovery and analysis of small, non-coding RNAs has been vastly facilitated by NGS technologies. Small RNA sequencing allows detection of all known and novel sequences, including the length and sequence variations, or isomirs, seen in microRNAs.

At ACGT, Inc., we use primarily the TruSeq Small RNA Sample Prep Kits from Illumina, which provide 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. Additional kits from other providers may be utilized at the customer’s request.

Table 2. Transcriptome Size vs Amount of Coverage

* 7 lanes is maximum per 1 flow cell