Impact of Sample Age on RNA Sequencing Metrics from FFPE Tissues Using Ion Torrent GenexusTM Integrated Sequencer
Laboratory Genetics and Genomics
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Primary Categories:
- Laboratory Genetics
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Secondary Categories:
- Laboratory Genetics
Introduction:
RNA fusions have important diagnostic, prognostic, and therapeutic implications in several solid tumor types. Next generation sequencing’s ability to assess a broad range of fusions simultaneously make it ideal for the detection of these fusions and Formalin-fixed paraffin-embedded (FFPE) tissues are the prime specimens for molecular profiling in solid tumors. Given the rapid evolution of tumor RNA profiling, archival FFPE specimens are often pulled and rerun on newer or broader panels in patients with progression of their disease. Compared to DNA, RNA is generally more prone to degradation in preserved FFPE tissue, leading to uncertainty regarding robustness of RNA sequencing from archived FFPE tissues. This study analyzes several RNA sequencing metrics on the new Ion Torrent platform, the Genexus Integrated Sequencer to investigate the impact of sample age on RNA concentration, quality and fusion markers detected in RNA extracted from the archived FFPE slides.
Methods:
RNA was manually extracted from FFPE slides, post deparaffinization, from 89 surgical pathology blocks archived from 2019-2023 using the Qiagen AllPrep DNA/RNA FFPE kit. RNA concentrations were determined by A260 on Nanodrop as well as by Qubit RNA high-sensitivity Assay kit. Library preparation, templating and sequencing was performed using Oncomine Precision Assay GX on the Genexus integrated sequencer. Three specimens failed the internal QC and were excluded from the final analysis. Reference genome hg19 was used for alignment. Data was analyzed by both manual curation and the default Oncomine extended 6.8 filters.
Results:
RNA metrics among all samples tested yielded wide distribution: total reads 662,424 ± 352,669, mapped reads 172,064 ±97,566, and mean read length 76.8 ± 17.5 bp. Whereas RNA quantity varied widely across individual samples and year, NGS metrics associated with RNA quality, namely, total RNA reads, mapped RNA reads, and mean RNA read length – were significantly lower in older samples compared to more recent ones (p< 0.001) regardless of RNA concentration. RNA concentrations of older samples, even when sufficient, is less indicative of sequencing success than sample age and should notbe a sole metric for assessment.
Conclusion:
RNA extracted from archived FFPE tissues exhibits considerable variability in sequencing metrics, with notable degradation and poorer NGS performance over time. The analysis revealed significant differences in total reads, mapped reads, mean read length with increasing time independent of RNA concentration. This suggests that the year of block preparation impacts the integrity and quality of RNA, highlighting the importance of establishing cutoffs and optimizing RNA sequencing protocols for older FFPE samples to ensure accurate and high-quality RNA profiling.
RNA fusions have important diagnostic, prognostic, and therapeutic implications in several solid tumor types. Next generation sequencing’s ability to assess a broad range of fusions simultaneously make it ideal for the detection of these fusions and Formalin-fixed paraffin-embedded (FFPE) tissues are the prime specimens for molecular profiling in solid tumors. Given the rapid evolution of tumor RNA profiling, archival FFPE specimens are often pulled and rerun on newer or broader panels in patients with progression of their disease. Compared to DNA, RNA is generally more prone to degradation in preserved FFPE tissue, leading to uncertainty regarding robustness of RNA sequencing from archived FFPE tissues. This study analyzes several RNA sequencing metrics on the new Ion Torrent platform, the Genexus Integrated Sequencer to investigate the impact of sample age on RNA concentration, quality and fusion markers detected in RNA extracted from the archived FFPE slides.
Methods:
RNA was manually extracted from FFPE slides, post deparaffinization, from 89 surgical pathology blocks archived from 2019-2023 using the Qiagen AllPrep DNA/RNA FFPE kit. RNA concentrations were determined by A260 on Nanodrop as well as by Qubit RNA high-sensitivity Assay kit. Library preparation, templating and sequencing was performed using Oncomine Precision Assay GX on the Genexus integrated sequencer. Three specimens failed the internal QC and were excluded from the final analysis. Reference genome hg19 was used for alignment. Data was analyzed by both manual curation and the default Oncomine extended 6.8 filters.
Results:
RNA metrics among all samples tested yielded wide distribution: total reads 662,424 ± 352,669, mapped reads 172,064 ±97,566, and mean read length 76.8 ± 17.5 bp. Whereas RNA quantity varied widely across individual samples and year, NGS metrics associated with RNA quality, namely, total RNA reads, mapped RNA reads, and mean RNA read length – were significantly lower in older samples compared to more recent ones (p< 0.001) regardless of RNA concentration. RNA concentrations of older samples, even when sufficient, is less indicative of sequencing success than sample age and should notbe a sole metric for assessment.
Conclusion:
RNA extracted from archived FFPE tissues exhibits considerable variability in sequencing metrics, with notable degradation and poorer NGS performance over time. The analysis revealed significant differences in total reads, mapped reads, mean read length with increasing time independent of RNA concentration. This suggests that the year of block preparation impacts the integrity and quality of RNA, highlighting the importance of establishing cutoffs and optimizing RNA sequencing protocols for older FFPE samples to ensure accurate and high-quality RNA profiling.