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Significant and pervasive effects of RNA degradation on Nanopore direct RNA sequencing

ABSTRACT: Abstract Oxford Nanopore direct RNA sequencing (DRS) is capable of sequencing complete RNA molecules and accurately measuring gene and isoform expression. However, as DRS is designed to profile intact RNA, expression quantification may be more heavily dependent upon RNA integrity than alternate RNA-seq methodologies. It is currently unclear how RNA degradation impacts DRS or if it can be corrected for. To assess the impact of RNA integrity on DRS we performed a degradation time-series using SH-SY5Y neuroblastoma cells. Our results demonstrate that degradation is a significant and pervasive factor that can bias DRS measurements, including a reduction in library complexity resulting in an overrepresentation of short genes and isoforms. Degradation also biases differential expression analyses; however, we find explicit correction can almost fully recover meaningful biological signal. In addition, DRS provided less biased profiling of partially degraded samples than nanopore cDNA-PCR sequencing. Overall, we find samples with RIN > 9.5 can be treated as undegraded and samples with RIN > 7 can be utilised for DRS with appropriate correction. These results establish the suitability of DRS for a wide range of samples, including partially degraded in-vivo clinical and post-mortem samples, whilst limiting the confounding effect of degradation on expression quantification.

PROVIDER: EGAS00001006542 | EGA |

REPOSITORIES: EGA

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Project description:Achieved biospecimens annotated with patient clinical characteristics are unique resources for translational research. However, RNA extracted from the achieved tissues is often degraded. RNA degradation can have a significant impact on the measure of transcript abundance that can lead to an increase rate of erroneous differentially expressed genes. Here, we are presenting the transcript integrity number (TIN) algorithm to measure the RNA degradation at transcript level. When applied to RNA-seq datasets generated from human brain Glioblastome cell line, human peripheral blood mononuclear cells, and metastatic castration resistant prostate cancer (mCRPC) clinical tissues, TIN provided a more reliable and more sensitive measure of RNA degradation than RIN, as demonstrated by much higher concordance with the RNA fragment size estimated from read pairs. More importantly, when comparing 10 mCRPC samples with lower RNA quality to another 10 samples with higher RNA quality, we demonstrated that calibrating gene quantification with TIN scores could mitigate RNA degradation effects and greatly improve gene expression analysis. The detected differentially expressed genes before TIN correction were predominantly ribosomal genes. However, when we adjusted gene quantifications with the corresponding TIN scores, we found differentially expressed genes were highly enriched in prostate cancer specific pathways. When further evaluating the performance of TIN correction using synthetic spike-in transcripts with predetermined abundance in RNA-seq data generated from Sequencing Control Consortium (SEQC), we found TIN adjustment had a better control of false positives and false negatives (sensitivity = 0.89, specificity = 0.91), as compared to gene expression analysis results without TIN correction (sensitivity =0.98, specificity = 0.50). RNA sequencing of 20 bone-metastatic castration resistant prostate cancer (mCRPC) using Illumina HiSeq 2500. Out of 20 mCRPC samples, 10 samples have relative low RNA integrity and another 10 samples have relative higher RNA integrity as measured by Agilent RIN score.

Project description:To determine the effect of RNA degradation on miRNA expression profiling. To mimic total RNA degradation in snap-frozen livers and duodenums from mice (wild type female, C57/Bl6) tissues were stored in a -80Â°C freezer and then transferred to dry ice. Each frozen tissue was sliced into five identical pieces which then were transferred into eppendorf tubes. At time point zero (T0), all samples were placed on ice and total RNA was extracted immediately from (T0) and at later time points [30 min (T30), 60 min (T60), 120 min (T120) and 240 min (T240)]. RNA integrities were assessed using the Agilent Bioanalyzer 2100 which calculates RIN values of assayed RNAs. We found that at T0, the RNA integrity for both liver and duodenum was above 7, indicating good quality total RNA. However, 30 min on ice was sufficient to reduce RNA integrity, indicated by the decrease in RINs. These findings indicate that RNA degradation can take place in defrosted tissue at low temperature (i.e. on ice). For comparison, we assessed the extent to which RNA integrity is preserved in freshly harvested tissues when tissue processing is delayed. Liver and duodenum samples were collected from mice and either processed immediately or maintained on ice, as described above. Bioanalyzer electropherograms of the liver samples did not indicate any significant total RNA degradation even when samples remained on ice for up to 4 hrs. By contrast, duodenal samples (which are rich in RNases) do show high susceptibility to degradation similar to the snap-frozen defrosted material. These findings suggest that tissues such as duodenum or pancreas should either be processed immediately or snap-frozen and processed individually. To assess to which extent total RNA degradation affects miRNA expression profiles, we hybridized to the miCHIP microarray total RNAs extracted from freshly harvested livers and duodenums liver and from snap frozen livers and duodenums. Hierarchical clustering (HCL), by Pearson correlation, was used to cluster samples based on their miRNA expression profiles. Our analysis shows that miRNAs extracted from freshly harvested liver is less degraded (matrix plot analyses). Consistent with high level of RNases present in duodenum, miRNAs from freshly harvested duodenum are extensively degraded. Based on these data, we conclude that samples with low RIN values (less than 7) do not merit analysis on miRNA arrays.

Project description:Purpose: Popular methods for library preparation in RNA-seq such as Illumina TruSeq® RNA v2 kit use a poly-A pulldown strategy. Such methods can cause loss of coverage at the 5’ end of genes, impacting the ability to detect fusions when used on degraded samples. The goal of this study was to quantify the effects RNA degradation has on fusion detection when using poly-A selected mRNA and to identify the variables involved in this process Methods: Total RNA was extracted from solid tumor tissue and whole blood using the Qiagen® miRNeasy Micro and Mini kits, respectively. The KU812 cell line was purchased from Sigma-Aldrich (St. Louis, MO) and UHR (Universal Human Reference RNA) was purchased from Agilent (Santa Clara, CA). UHR is a mixture of cell lines derived from breast adenocarcinoma, hepatoblastoma, cervix adenocarcinoma, testis embryonal carcinoma, gliobastoma, melanoma, liposarcoma, histiocytic lymphoma, lymphoblastic leukemia and plasmocytoma. For Degradation experiments, two micrograms of human universal reference RNA (UHR) (Agilent Technologies, Santa Clara, CA) and 1ug of RNA extracted from KU812 cell line (purchased from ATCC) were degraded at 74oC from 1 to 11 minutes in 1 minute intervals, using the NEBNext® Magnesium RNA Fragmentation Module Kit (NEB, Ipswich, MA). RNA was then purified and concentrated with RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA). Results: In this study, we designed experiments using artificially degraded RNA from cell lines as well as naturally degraded RNA from tissue samples to quantify the effect RNA degradation has on fusion detection when using poly-A selected RNA libraries We found that both the RNA degradation level and the distance from the 3’ end of a gene, negatively impact the read coverage profile in RNA-seq. Furthermore, the median transcript coverage decreases exponentially as a function of the distance from the 3’ end and there is a linear relationship between the coverage decay rate and the RNA integrity number (RIN). Conclusions: we found that when using poly-A pulldown techniques for library preparation in RNA-seq, the fusion sensitivity is negatively impacted by both sample degradation and distance of the fusion breakpoint from the 3’ end and developed graphs that show such effect. Such graphs can be useful in assessing the fusion sensitivity of RNA-seq in both research and clinical settings

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Significant and pervasive effects of RNA degradation on Nanopore direct RNA sequencing

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