Most Vertebrate Viral Proteomesin a Single Assay

Technical Details

The VirScan PhIP-Seq library is designed to target >68,000 annotated viral protein sequences from most known proteins from vertebrate, mosquito-borne, and tick-borne viral genomes according to the UniProt and RefSeq databases in 2017. VirScan contains an E. coli codon-optimized library consisting of >480,000 oligonucleotides encoding 62-mer amino acid peptide inserts with 14-mer amino acid adjacent overlaps.

The oligonucleotides library was synthesized on a releasable microarray, PCR amplified using appended adapter sequences, and cloned en masse into a modified T7-Select vector. This vector displays a library peptide as a fusion to the T7 phage capsid protein. The library was then packaged in vitro and expanded. The quality of library inserts was assessed by Illumina sequencing.

Screens are performed in PBS which contains phage library at an average of 105 excess clones per unique phage in the input library. The mixture is rotated overnight to allow antibodies to bind any phage-displayed peptide targets. The next day, a Protein A/G coated magnetic bead slurry is added and rotated for an additional 4 hours to capture all IgG. A set of independent Protein A/G beads-only mock-immunoprecipitation buffer-alone samples are included as negative controls. All protein A/G beads are then washed and resuspended with a Herculase II Fusion Polymerase master mix and amplified with PCR. Multiplex barcodes and sequencing adapters are then incorporated during a subsequent PCR reaction. Barcoded samples are pooled and sequenced using Illumina instruments to obtain paired-end reads of the amplified phage inserts.

Service Details and Data Deliverables

A VirScan service involves case and control serum or plasma samples. These undergo a protein A/G pulldown assay, PCR amplification, and next-generation sequencing. Raw sequence data are run through a normalization and quantitation pipeline. Raw pipeline counts outputs are then provided to customers alongside normalized hit calls data for all individual 62mer peptides.

Sample Requirements

Serum or plasma20 μL aliquot per sample

Dilute serum or plasma2.5 μL aliquot in 247.5 μL PBS per sample

Cerebrospinal fluid (CSF)250 μL aliquot per sample

Other antibodies (IgG monoclonals, B cell supernatants, etc)250 μL aliquot per sample at 0.1 mg/mL concentration

12 sample minimumSold in multiples of x12 samples (i.e., x12, x24, x36, 48x – full rows of a standard 96-well plate), and any number after 48 samples

Cohort balancing (studies bigger > 336 samples)

Individual PhIP-Seq studies are prepared in x96 well plates using aliquots of our phage library; each study requires x48 controls pooled plasma, known polyclonal, and protein A/G beads-only internal control samples per sequencer run (provided free-of-charge). Data are most reproducible within a single sequencer run. Sequencer runs are currently limited to 4x 96-well plates (336 experimental samples + x48 controls per run).

Sample Shipping and Sample Return

We will include shipping details in your quote – you must cover the cost of shipping samples to CDI Labs. Typically, after you receive your report, CDI Labs keeps the remaining samples for two months and then disposes of them. When shipping to us, please let us know if you want the remaining samples returned after the study is complete. Return shipping will be charged.