The cDNA-PCR Sequencing Kit (SQK-PCS111) and PCR-cDNA Barcoding Kit (SQK-PCB111.24) are recommended for users who want to maximise read count output and/or have limited amounts of input RNA. We also recommend this kit for users wanting to explore isoforms, splice variants and fusion transcripts using full-length cDNAs.

However, on a rare occasion, we have observed a biased amplification of a set of short (170-200 bp) technical artefactual sequences that may reduce yields from the target sample. If pronounced, the artefact can be detected on the Agilent Bioanalyzer (Figure 1). We found an additional AMPure XP bead size selection can be performed to effectively remove the artefact from the library before reamplifying the target transcriptome with additional PCR cycles. We recommend reamplifying the size selected product until at least 15 ng is generated, which is sufficient to load 25 fmol of library onto the flow cell, as recommended in the protocol.

In the example below, we demonstrate our recommendations using a target sample which was sufficiently reamplified with four additional cycles. However, please note that the number of cycles are likely to vary between samples based on the input mass as well as the ratio of artefact to target sample. Therefore, an initial six cycles are recommended to ensure successful recovery.

Figure 1. Agilent 2100 Bioanalyzer image of SQK-PCS111 sample was found to contain considerable amounts of ~175 bp PCR artefact with limited amplification of the target transcriptome (green). Size selection and reamplification of the sample (yellow) removed the artefact and enriched the target sample.

Figure 2. Read length profiles for SQK-PCS111 libraries. The upper panel shows a typical library (using UHRR as the template); while this does contain some artefact (blue), most of the time is spent sequencing the target transcriptome (green). The centre panel shows an SQK-PCS111 library with highly abundant artefact which reduces the number of reads from the transcriptome. The bottom panel shows the same library after size selection and reamplification to rescue the library.

To assess the impact of the treatment, we added quantitative Spike-in RNA Variants SIRV-Set 3 (Lexogen) to a Universal Human Reference RNA (UHRR) sample and prepared a library using cDNA-PCR Sequencing Kit protocol (SQK-PCS111). The quantitative panel includes 92 unique transcripts designed by the External RNA Controls Consortium (ERCC) and have a dynamic concentration range of six orders of magnitude. The treated library had a Spearman’s rank coefficient > 0.92 with the known spike-in concentrations, indicating that the additional steps do not introduce a significant bias.

Rescue method:

In the cDNA-PCR Sequencing (SQK-PCS111) protocol and PCR-cDNA Barcoding Kit (SQK-PCB111.24) protocol, we recommend analysing your amplified sample using an Agilent Bioanalyzer or equivalent as a QC check post-PCR clean-up. If your amplified cDNA shows PCR artefact, similar to that of Figure 1, use the following steps to recover the sample after the QC check, before the “Adapter addition” step of the protocol:

1. Add 40 µl of Elution Buffer (EB) to 10 µl of the eluate sample, for a total of 50 µl.

2. Resuspend the AMPure XP beads by vortexing.

3. Add 30 µl of resuspended AMPure XP beads to the diluted sample.

4. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.

5. Prepare 500 µl of fresh 70% ethanol in nuclease-free water.

6. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

7. Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

8. Repeat the previous step.

9. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

10. Remove the tube from the magnetic rack and resuspend the pellet in 20 µl of Elution Buffer (EB).

11. Repeat steps 1-22 of the "Selecting for full-length transcripts by PCR", modifying the PCR conditions to complete six cycles instead of 14 cycles.

12. Analyse 1 µl of your reamplified cDNA using a Qubit fluorometer and Agilent Bioanalyzer (or equivalent) to check whether the artefact has been removed and that there is enough sample to sequence 25 fmols on a flow cell.

If the artefact has not been removed and/or you do not have enough sample for sequencing, repeat the above steps again.