Prior to RNA/cDNA sequencing, it is common practice to perform quality control on samples using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN). The RIN assigns a measurement of the level of degradation of the RNA in the extract from 1 to 10, with 10 being the least degraded. For more information, go to https://www.agilent.com/cs/library/applications/5989-1165EN.pdf .

It is recommended that RNA extracts have an RIN of 7 or higher before proceeding with library preparation for nanopore sequencing. We took RNA extracts of a human lymphoblastoid cell line (COLO829 BL) and quantified using the Qubit™ fluorometer. The RIN was determined using the Agilent 2100 Bioanalyzer before samples of seven different RIN values were taken forward and libraries prepared using the PCR-cDNA Sequencing Kit (SQK-PCS111), starting from 200 ng of total RNA with 14 PCR cycles and sequenced on PromethION. Prior to library preparation, 450 pg of SIRV-Set 3 was added to each sample (this is expected to result in ~5% of reads being attributed to the spike-in). The sequencing runs yielded >50 million reads for each of the libraries in 24 hours.

Figure 1. The proportion of reads aligned between the SIRV-Set 3 and target organism (human) for samples with different RIN values. In the samples with high RIN values (10 – 7.8), the expected ratio of reads aligning to the target organism and SIRV-Set 3 is observed with ~5% of reads aligning to the spike-in and ~95% of reads aligning to the target genome. For the sample with the lowest RIN of 3.5, ~22% of reads aligned to the spike-in, suggesting that little viable target template is present in the sample. The Spearman’s rank correlation coefficient (rho) for the ERCC alignments were >0.97 in each case, suggesting that there is very little bias in the library preparation and sequencing.

Figure 2. The length distribution of reads that align to the target organism (human) or the spike-in in samples with different RIN values. As the RIN value decreases, it is observed that the read length distribution is skewed towards shorter molecules and the read N50 decreases, indicating that the RNA in the sample is progressively more degraded.

Figure 3. The read length distribution of ERCC aligned reads. The observed lengths of the reads that aligned to the ERCC panel appeared to be independent of the length of the reads that aligned to the target genome, with the majority of reads covering almost the entire transcript. This is expected as the SQK-PCS111 kit enriches for full-length RNA template molecules. However, this suggests that the shorter reads observed where the RIN value is low is not due to bias in the library prep, but is due to the level of degradation that has occurred in the sample.

Figure 4. The library yield for samples with different RIN values. It is observed that yield from the library decreases as the RIN of the target sample drops below 7, generating ~50 ng of library (post clean-up), which is sufficient for the recommended flow cell loading mass (15-50 ng).

Change log

Version	Change note
v2, 3rd March 2022	Updated with new data relevant for the new SQK-PCS111, using COLO829 BL cells grown in culture
v1	Initial protocol publication