Total RNA or individual populations of RNA, such as mRNA or small RNA are captured and converted to cDNA and subjected to NGS for analysis. Common rRNA and tRNA as well as abundant mRNA transcripts such as globin can be targeted for depletion in order to focus sequencing throughput on RNA of interest.

Applications:

• Discover novel transcripts, identify intron/exon boundaries or discover alternative splicing isoforms
• Identify specific strand information, genefusions or mutations/SNPs
• Observe alterations in gene expression or mutation cancer
• Determine host/pathogen interactions
• Characterize bacterial pathogenesis, antimicrobial resistance or persistence
• Identify viral quasi-species or latency activation

At Biomiga, we understand that the quality of RNA going into next generation sequencing is directly related to the quality of sequence data coming out. That is why quality metrics are captured at vital points:

• Post-extraction: RNA is analyzed to ensure it is of sufficient integrity and quantity for optimal post-library preparation chemistries
• Post-library preparation: Fragment size and quantity is again checked to ensure optimal loading onto the Illumina flow cell
• NGS run: The quality (Q) scores, along with cluster densities, percentage of reads passing filter, number of reads generated and average length of reads

Biomiga 'targeted sequencing services will guide you all the way from project inception to high-quality results. Our expert personnel and state-of-the-art equipment allow for high-quality data and fast turnaround times. Sequencing is performed on an Illumina HiSeq 2500 utilizing V4 chemistry, which allows you to receive maximum value for your samples. Results are delivered via secure cloud drive or delivery via external hard drive is available.

• Fast turnaround times
• Highly experienced personnel to guide you from design to data
• Data analysis report that includes QC metrics, alignment files and variances
• Custom bioinformatics available and free basic bioinformatics available through Illumina Basespace
• A dedication to quality

Product Overview

The sequencing of RNA is available as both targeted and non-targeted approaches. For a non-targeted approach, the total RNA, or whole transcriptome, from a sample can be converted to cDNA, fragmented, end-repaired and adapter-ligated for NGS. This approach captures all RNA (mRNA, rRNA, tRNA and miRNA). However, if the questions being asked are more circumspect, then targeted or enrichment approaches are available to specifically pull out mRNA from the RNA pool by use of poly T probes, or alternatively deplete the abundant rRNA, tRNA or globin RNA with probes specific for those species can enrich the sample for the remaining RNA of interest and provide better sequence coverage.

For further focus, targeted strategies for genes of interest involve the use of the user-designed specific primers that can amplify a selected set of genes, such as those for a particular metabolic pathway. These primers are designed to also contain the sequences for direct NGS applications.

NGS library preparation strategies are available that contain adapters with a chemical modification that specifically targets the 3 end of miRNA allowing for their specific amplification and sequencing of this species from total RNA.

Since NGS is still a single-stranded DNA-based application, RNA has to be converted to cDNA prior to NGS. However, molecular methods allow for sequencing only the cDNA strand that represents the original RNA single stranded molecule by the incorporation of dUTP into the second strand during cDNA synthesis. This strand is then destroyed via Uracil-DNA-Glycosylase prior to final amplification and is not sequenced.

The sequence exploration of RNA with its targeting and enrichment strategies, dynamic range and quantitative information can be a highly valuable tool for discovering:

• Intron/exon boundaries
• Alternative gene splicing/isoforms
• Novel miRNA and mRNA transcripts
• Precise strand information
• Gene fusion
• Mutations/SNPs
• Changes in gene expression
• New gene sequence annotation
• Cancer alterations in gene expression
• Host/pathogen interactions
• Viral quasi-species
• Viral latency activation
• Bacterial pathogenesis, resistance and persistence