Webinar: Total RNA sequencing of liquid biopsies

RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare.

In this webinar, Jo Vandesompele, PhD - CSO at Biogazelle, discussed the performance of the SMARTer Stranded Total RNA-Seq method and showed:

• Its application to various biofluids and extracellular vesicles
• Accuracy, precision and sensitivity of the method
• Diversity of RNA biotypes identified and their heterogeneity among different biofluids
• Performance data with other sample types: FFPE tissue and single cells

The SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian is used to generate strand-specific RNA-seq libraries for Illumina sequencing from 250 pg–10 ng inputs of purified total RNA.  This kit incorporates Takara Bio’s proprietary SMART (Switching Mechanism at the 5’ end of RNA Template) technology and includes refinements to the SMARTer method for stranded RNA-seq that simplify the library preparation workflow and improve sequencing performance. This method was developed to work with either high- or low-quality total RNA, does not require additional rRNA removal methods or kits, and produces sequencing libraries that retain strand-of-origin information. The integrated removal of cDNAs derived from rRNA—typically present in high abundance following cDNA synthesis from total RNA inputs—makes the workflow extremely sensitive, yielding data that is highly reproducible with low mapping to rRNA. The new library design featured in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian improves sequencing performance compared to the original SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian, particularly for NextSeq® and MiniSeq™ instruments carrying the two-channel SBS technology. This kit includes the Indexing Primer Set HT for Illumina v2; for your convenience, we also offer the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian Components (Cat. #s 634418 and 634419) without indexing primers.

Schematic of sequencing libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Structural features of final libraries generated with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. The adapters added using 5' PCR Primer HT and 3' PCR Primer HT contain sequences allowing clustering on any Illumina® flow cell (P7 shown in light blue, P5 shown in red), Illumina TruSeq® HT indexes (Index 1 [i7] sequence shown in orange, and Index 2 [i5] sequence shown in yellow), as well as the regions recognized by sequencing primers Read Primer 2 (Read 2, purple) and Read Primer 1 (Read 1, green). Read 1 generates sequences antisense to the original RNA, while Read 2 yields sequences sense to the original RNA (orientation of original RNA denoted by 5' and 3' in dark blue). The first three nucleotides of the second sequencing read (Read 2) are derived from the Pico v2 SMART Adapter (shown as Xs). These three nucleotides must be trimmed prior to mapping if performing paired-end sequencing.

Improved sequencing performance with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved pass-filter rates (%PF) with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. Libraries generated with the Pico v1 or Pico v2 kits were pooled and run on NextSeq 500 or MiniSeq instruments, as indicated. For each graph, blue boxplots indicate the distribution of cluster densities for unfiltered (i.e., raw) reads, while the green boxplots indicate the distribution of cluster densities for reads that passed filtering. Quantities of reads passing filter (in millions) and %PF values for each sequencing run are included above each graph. The expected number of reads passing filter according to Illumina specifications was 130 million reads for runs on the NextSeq and 25 million reads for runs on the MiniSeq. Proportions of reads that aligned to PhiX sequences ranged from 0.5% to 1.15% for all sequencing runs. As indicated in the graphs, libraries generated with the Pico v2 kit achieved higher %PF values for both Illumina platforms relative to libraries generated with the Pico v1 kit, and yielded quantities of reads passing filter that greatly exceeded the Illumina specifications.

Workflow for SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Schematic of technology in the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. SMART technology is used in this ligation-free protocol to preserve strand-of-origin information. Random priming (represented as the green N6 Primer) allows the generation of cDNA from all RNA fragments in the sample, including rRNA. When the SMARTScribe Reverse Transcriptase (RT) reaches the 5' end of the RNA fragment, the enzyme’s terminal transferase activity adds a few non-templated nucleotides to the 3' end of the cDNA (shown as Xs). The carefully designed Pico v2 SMART Adapter (included in the SMART TSO Mix v2) base-pairs with the non-templated nucleotide stretch, creating an extended template to enable the RT to continue replicating to the end of the oligonucleotide. The resulting cDNA contains sequences derived from the random primer and the Pico v2 SMART Adapter used in the reverse transcription reaction. In the next step, a first round of PCR amplification (PCR1) adds full-length Illumina adapters, including barcodes. The 5' PCR Primer binds to the Pico v2 SMART Adapter sequence (light purple), while the 3' PCR Primer binds to sequence associated with the random primer (green). The ribosomal cDNA (originating from rRNA) is then cleaved by ZapR v2 in the presence of the mammalian-specific R-Probes v2. This process leaves the library fragments originating from non-rRNA molecules untouched, with priming sites available on both 5' and 3' ends for further PCR amplification. These fragments are enriched via a second round of PCR amplification (PCR2) using primers universal to all libraries. The final library contains sequences allowing clustering on any Illumina flow cell.

Improved sequencing metrics with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian

Improved sensitivity and reproducibility with the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian. Sequencing libraries were generated from 1 ng and 10 ng inputs of total RNA extracted from human lung FFPE tissue using both the Pico v1 and Pico v2 kits, and sequenced on a NextSeq 500 instrument. Panel A. Sequencing metrics for libraries generated from 1 ng or 10 ng inputs using each kit. For both input amounts, the Pico v2 kit resulted in greater library yields, a lower proportion of reads mapping to rRNA and mtRNA, and a lower duplicate rate. For the 1 ng input, sequencing data from the Pico v2 library also identified thousands more transcripts than sequencing data from the Pico v1 library, indicating a higher sensitivity for Pico v2. Panel B. Comparison of transcript expression levels across input amounts. Higher reproducibility was observed between 1 ng and 10 ng inputs for data generated with the Pico v2 kit vs. data generated using the Pico v1 kit. FPKM values are shown on a Log10 scale. Transcripts represented in only one library can be seen along the X- and Y-axes of the scatter plots.

SeqAmp CB PCR buffer improves bead-pellet formation

Improved bead-pellet formation with new SeqAmp CB PCR buffer. The PCR buffer included in the Pico v2 kit was re-formulated to allow for faster, tighter bead-pellet formation. Following magnetic separation for a fixed period, bead pellets formed in the new SeqAmp CB buffer (right) are tighter than those formed in the original PCR buffer (left). Tighter bead pellets tend to dry more evenly and are easier to resuspend than pellets that are broader and more diffuse.