Table 1

Potential solutions to overcome SARS-CoV-2 library preparation challenges

ChallengePotential causeSolution
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 inputInsufficient 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 beadsBeads 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 TapeStationInappropriate 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 PCRAdapters 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 concentrationTo 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.comLow internet bandwidth while using BaseSpace sequence hub.We used off-instrument implementation of Local Run Manager:
Quality control (QC) metrics of sequencing runDue 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:
  • CZB, Chan Zuckerberg Biohub; CZI, Chan Zuckerberg Initiative; SPRI, Solid-phase reversible immobilization.