Potential solutions to overcome SARS-CoV-2 library preparation challenges
| Challenge | Potential cause | Solution |
| Global supply chain issues (a complex network of manufacturing, processing and resource delivery) | Global shortage due to worldwide increase in sequencing kits/reagents for surveillance, plus pandemic-struck shipping causing overall supply chain issues. | We received a few supplies and reagents initially for the sequencing run from our international collaborators (CZB/CZI). Now, we are ordering even more in advance, or trying to order straight from the vendor instead of the distributor (less likely). |
| DNA input | Insufficient input of DNA (10 ng) was used per reaction. | Increased the input of DNA from 10 ng to 50 ng. Reduced barcoding PCR cycles from 14 to 12. |
| Very low yield of DNA even after concentrating them via beads | Beads were not resuspended properly, not adjusted to room temperature or accurate volume was not picked. | We completed further optimisation and quality control of the different SPRI bead ratios using ladders (untreated, 1.0×, 0.8×, 0.75×, 0.7×, 0.65× and 0.6×). |
| Adapter dimers issues on diagnostic PCR gel and TapeStation | Inappropriate fragmentation time of library preparation and/or SPRI beads clean up. | For the elimination of adapter dimers, we performed two SPRI clean-ups at 0.8× ratio after the barcoding PCR step. We also reduced the fragmentation time from 5 min to 3 min for library preparation. |
| Bands were not observed for diagnostic PCR | Adapters were not added, too low SPRI beads ratio, low concentration of samples on Qubit. | Adapters were added separately and not as part of a master mix, the corrected volume of SPRI beads was added during the clean-up. |
| Initial loading concentration | To avoid overloading, iSeq concentration range was set to 75 pM and 120 pM. | Set to optimal loading concentration of 100 pM of pooled library. |
| Problem in using BaseSpace sequence hub https://basespace.illumina.com | Low internet bandwidth while using BaseSpace sequence hub. | We used off-instrument implementation of Local Run Manager: https://support.illumina.com/sequencing/sequencing_software/local-run-manager/downloads.html |
| Quality control (QC) metrics of sequencing run | Due to internet bandwidth, we did not run QC online using BaseSpace. | QC metrics of sequencing run was done locally using Sequence Analysis Viewer (SAV) software: https://support.illumina.com/sequencing/sequencing_software/sequencing_analysis_viewer_sav/downloads.html |
CZB, Chan Zuckerberg Biohub; CZI, Chan Zuckerberg Initiative; SPRI, Solid-phase reversible immobilization.