As researchers delve deeper into the human genome, demand for highly sensitive and reproducible RNA-seq is growing. Next-generation sequencing (NGS) applications offer diverse and detailed analysis for these studies, but manual library preparation is repetitive, tedious, and prone to human error and variability. In contrast to manual preparation methods, the Apollo system brings the benefits of high-throughput automation to the equation. By combining the industry-leading sensitivity of SMART-Seq v4 (SSv4) ultra-low-input cDNA synthesis with the consistency of the Apollo instrument, we developed an automated workflow for ultra-low-input and single-cell RNA-seq, yielding comparable, if not better, results for cDNA synthesis versus manual protocols, while also decreasing hands-on time and variability.
- SmartChip Real-Time PCR System introduction
- ICELL8 introduction
- Apollo library prep system introduction
Automating sensitive SMART-Seq v4 chemistry for reliable, high-throughput results on the Apollo system
To analyze the use of SSv4 chemistry on the automated system, cDNA amplification for library preparation was performed on three different Apollo instruments with 48 samples per run (40 with Control Total RNA and eight no-RNA controls). For each run, four bench controls (three with Control Total RNA and one no-RNA control) were performed manually using the same master mixes and sharing the same thermal cycler as the automated samples.
Similar profiles and yields for automated and manual cDNA amplification
Amplified cDNA was analyzed with an Agilent 2100 Bioanalyzer and Agilent's High Sensitivity DNA Kit (Figure 1). Results showed similar amplified cDNA profiles between automated replicates, demonstrating consistency between runs. These automated replicates also showed similar profiles when compared to the manually generated cDNA, indicating that the SSv4 technology performs just as well under these time-saving, high-throughput conditions as it does with manual benchtop workflows.
The yield was calculated for the region from 300–9,000 bp and then averaged across the three Apollo runs, with 40 samples each. The three sets of manual controls with three samples each were subjected to the same calculations. Overall yield was not significantly different between the two amplification methods (Table 1).
|Workflow||Number of samples||Average yield (ng)|
Table 1. Bioanalyzer quantification of automated replicates compared to manual samples.
Sequencing libraries generated using automated and manual amplification protocols show similar metrics and high correlations
A subset of the cDNAs amplified above (33 Apollo-generated samples and nine manually generated samples) were used in a manual Nextera® XT protocol for library preparation. The resulting libraries were sequenced on a NextSeq® instrument with 75-bp paired-end reads. Reads were trimmed and mapped to rRNA and the mitochondrial genome. Remaining unmapped reads were mapped to the mouse genome, producing uniquely mapped reads and percentage of reads that mapped to RefSeq annotations, including exons, introns, and intergenic regions. The number of transcripts identified in each library was determined by the number of transcripts with an FPKM ≥0.1 or FPKM ≥1. Sequencing metrics from each of these runs show similar results for libraries generated from cDNA amplified by either workflow (Table 2).
|Workflow||Number of samples||% rRNA||Number of transcripts
(FPKM > 0.1)
|Number of transcripts
(FPKM > 1)
|Range of Pearson correlations|
Table 2. Similar sequencing metrics between libraries generated from cDNA amplified by automated and manual protocols. FPKM = fragments per kb mapped.
Libraries generated from both types of samples showed high correlation between those from the manual workflow and those from the Apollo instrument (Figure 2), indicating that scaling up to a high-throughput protocol does not affect the capabilities of the SMART-Seq chemistry.
Automation of our sensitive SMART-Seq v4 technology for library preparation for ultra-low-input or single-cell RNA-seq on the Apollo system provides researchers with a walkaway solution to high-throughput cDNA synthesis and downstream NGS applications. This high-throughput system displays consistency between automated replicates and provides results that show a high similarity with those of manually generated libraries, without the tedious manual processing. Learn more about the Apollo system and how it increases reproducibility while minimizing sample loss by reading our in-tip bead separation tech note.
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