We have now received several hundred de-identified clinical samples from the Dominican Republic and Colombia. Unfortunately, they all have extremely low viral lows (Ct values > 38 in the few that are positive), so we’re continuing our technology development. We are following a couple of different strategies:
- Amplicon-based amplification of cDNA to create enough starting material for sequencing. We are using different protocols including an amplicon scheme developed in our lab, in Dave & Shelby O’Connor’s lab in Madison, and Greg Ebel’s lab in Fort Collins.
- AmpliSeq. We are also trialing the AmpliSeq protocol from Thermo Fisher that Ian Goodfellow and colleagues used successfully in Sierra Leone during the Ebola outbreak (the Ebola version that is…). Using the AmpliSeq panel requires using IonTorrent (we normally use Illumina) and Thermo Fisher has kindly provided a full setup including the S5, that we’re currently testing out.
- Hybrid-select based enrichment. We are also in the process of getting hybrid select methods setup, which will allow us to enrich sequencing libraries for viral material.
We are hopeful that at least one of these strategies will prove successful, and we will release our data here and on virological.org as it is generated.
For now, we are releasing another experiment (biological replicate) of the Malaysian strain P6-740 seed stock that we have used previously. Having independent replicates of the same seed stock will allow us (and others!) to fine-tune our computational pipelines and evaluate accuracy of intrahost diversity iSNV calling.
P6-740, positive control of Malaysian strain P6-740 passaged once on BHK-21 cells [20,729 ZIKV reads]
Total RNA was depleted for rRNA and sequenced on the Illumina MiSeq using previously published protocols with no specific amplification. A water-extraction control was also run. Filters based on the water control were used to remove contaminants from the samples and Kraken was used to do metagenomic analyses on both the raw and filtered reads. Human reads have been removed from all raw files using bmtagger and SNAP.
- Raw reads (depleted of human reads)
- Aligned reads (using Novoalign)
- Kraken metagenomic analysis (unfiltered)
- Kraken metagenomic analysis (filtered on water-only control)
- FastQC quality report
As always, please feel free to contact us directly if you have any questions or comments. We will continue to make data available as it is generated.