RNA sequencing (RNA-seq) is widely used to perform expression analysis of entire transcriptomes with high sensitivity and a wide dynamic range. Despite being a pivotal tool in biomarker discovery, RNA-seq can be challenging if the RNA samples have a low concentration or are degraded. This is common when working with patient-derived samples such as tissue biopsies, blood, or other biofluids.

The SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input Mammalian (referred to as “Pico v3”) is designed for efficient preparation of sequencing libraries from picogram amounts (10 ng–250 pg) of high- or low-quality total RNA (RIN 2–10, DV200 > 25%).

A random priming approach enables whole transcriptome profiling of coding and noncoding RNA biotypes (e.g. mRNA, lncRNAs, lincRNAs, etc.). Unlike a dT priming approach which relies on intact polyA tails, a random priming approach is also ideal for library preparation from degraded samples, including formalin-fixed paraffin-embedded (FFPE) tissue, laser capture microdissection (LCM) samples, and cell-free RNA (cfRNA) from biofluids, serum, plasma, and exosomes.

Using a combination of SMART^® (Switching Mechanism at 5’ end of RNA Template) technology with ZapR^® innovation for ribosomal cDNA depletion, Pico v3 is a complete solution for ribodepleted, strand-specific libraries with full gene body coverage for information on isoforms, fusions, etc. ZapR technology is a cas9-based post-library depletion technique to effectively deplete rRNA from picogram amounts of total RNA, which plays an essential role in achieving success with low-input amounts of total RNA. Competitor depletion technologies are limited to significantly higher nanogram inputs.

The workflow also incorporates unique molecular identifiers (UMIs) during reverse transcription to mitigate potential PCR errors and amplification biases, enabling quantitative analysis and accurate variant calling, especially for rare mutations.

Biomarker discovery also benefits from faster sequencing turnaround times. Singular Genomics has developed an innovative benchtop sequencer, the G4 Sequencing Platform, that leverages a 4-color rapid sequencing by synthesis (SBS) chemistry with advanced optics and fluidics engineering to provide single-day turnaround times across all applications. By combining fast run times and the ability to run up to 4 flow cells, with 16 independently addressable lanes, G4 delivers accuracy, speed, power, and unprecedented flexibility for a wide range of genomic applications.

Here we expand our existing Pico V3 kit—initially designed for Illumina^® sequencing technology—to work seamlessly with other sequencing platforms such as the G4. We show that total RNA libraries prepared with Pico v3 from 10 ng or 0.25 ng of Human Brain Total RNA Control yields similar sequencing metrics and data quality on the Singular Genomics G4 Sequencing Platform as on industry-standard sequencing platforms. These results show that researchers now have access to a fast, accurate, and cost-effective solution to power biomarker discovery from challenging and low input samples.

The SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Cat. #634485, #634486, #634487, #634488) was used to generate total RNA libraries from 10 ng or 0.25 ng of Human Brain Total RNA Control. Illumina libraries were prepared according to the user manual. This protocol was modified to prepare the Singular Genomics libraries. Singular UDI primers (#700,134; #700,135) were used in the second round of PCR amplification (PCR 2) to replace the Illumina adapters and add barcodes. Alternatively, Singular UDI primers may be used for the first round of PCR amplification (PCR 1) to add sequencing platform-specific adapters and barcodes. PCR 2 would then use universal S1/S2 primers (sequence information from Singular Genomics) for library amplification post-depletion.

For each sequencing platform, two library replicates (Rep1 and Rep2) were prepared for each of the two inputs (10 ng and 250 pg). All completed libraries were quantified using the Qubit 2.0 Fluorometer and Agilent TapeStation. The libraries were pooled at equimolar ratios before sequencing. Run format for Singular G4 sequencing was 2 x 76 + 12 bp dual indices. Flow cell type was F2, 200 cycle kit. Illumina libraries were sequenced on the NextSeq^®2000, P1 with a run format of 2 x 150 + 8 bp dual index. Sequences were trimmed to 76 bp for analysis. Data analysis was performed by downsampling both sets of sequencing data to 2 x 10⁶ reads. Data analysis was performed using Cogent™ NGS Analysis Pipeline v.2.0.

Similar library quality for RNA-seq

Preparing high-quality RNA-seq libraries is crucial for generating reliable RNA-seq data, especially in low-input total RNA-seq assays. The total RNA libraries prepared for the Singular Genomics G4 and the Illumina platform yielded similar profiles and size distributions, as shown by the TapeStation traces (Figure 1 and 2).

The TapeStation traces showed consistent library profiles with average sizes ranging from 379–391 bp. The Qubit concentration for the 10 ng Reps was 33.0–37.0 ng/μl for Illumina and 58.2–59.4 ng/μl for Singular, while the molarity was 128.53–146.37 nM for Illumina and 246.40–244.80 nM for Singular. 

For the 250 pg Reps, the Qubit concentration was 18.4–27.8 ng/μl for Illumina and 22.92–36.9 ng/μl for Singular, while the molarity was 71.85–107.73 nM for Illumina and 95.64–157.60 nM for Singular. For both low and high input samples, the Singular UDIs may require fewer PCR cycles to achieve necessary library yields.

Figure 1. NGS library profile for Illumina NextSeq2000.

Figure 2. NGS library profile for Singular Genomics G4. 

The Singular Genomics libraries show a small high molecular weight (HMW) peak, however this doesn’t affect sequencing results (Figure 3). This HMW peak can be reduced by decreasing the number of cycles for PCR 2 to 8 cycles for 10 ng RNA input and 12 cycles for 250 pg input.

Switch sequencers without affecting sequence quality

RNA-seq data generated on the Singular Genomics G4 Sequencing Platform and Illumina sequencing platform showed comparable performance. Read distribution (62–67% exons, 26–31% introns, 7% intergenic, 5% mitochondrial, 15–22% ribosomal), number of genes identified, UMIs counts, and strand specificity were nearly identical between the platforms (Figure 3). The Pearson correlation coefficients (>0.95) and Spearman correlation coefficients (>0.86) indicate a high level of transcript expression correlation between replicates and sequencing platforms (Figure 4).

Figure 3. Comparable transcript features detected for RNA-seq libraries sequenced with the G4 and NextSeq 2000 sequencers 

Figure 4. Excellent correlation of transcript abundance between Illumina and G4 libraries

Generating high-quality libraries from low-input samples can be challenging, especially when dealing with low-quality or degraded RNA. The SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian is ideally suited for such samples based on its random priming approach and leveraging the SMART technology integrated with the ZapR method for depleting rRNA from low input samples. Pico v3 kits solve many of the challenges posed by kits that require higher inputs, oligo(dT) primed approaches, or only end-counting methods.

Libraries can also be generated using SMART-Seq^® Total RNA Pico Input with UMIs (ZapR Mammalian) (Cat. # 634354, #634355, #634356), an updated version of Pico v3 which includes improved ZapR reagents designed for more efficient depletion of ribosomal RNA reads. Combining the power of the G4 Sequencing Platform with SMART-Seq technologies allows researchers to quickly achieve high-quality, reproducible detection of important genetic features from low input samples for biomarker discovery.