Immunology in 30 seconds... 

Immune challenge

Images: RSV: CDC/ Dr. Erskine Palmer -Public Health Image Library (PHIL), #276.

ebola: CDC/ Dr. Frederick A. Murphy - Public Health Image Library (PHIL),  #1833

cocksackie virus: Public Domain, wikimedia commons

Immunoglobulin

Heavy Chain

Light Chain

Hypervariable regions

IgG

Diversity...

5 heavy chain (γ, δ, α, μ and ε)

2 light chain(κ, λ)

 each with 3 hypervariable regions

e.g. 44 V 27D 6J  

VDJ recombination

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089068/

VDJ recombination + hypermutation = Tremendous Diversity

Recognition

Residues interact with 

target protein

Purpose of AbSeq: Identify these residues

Applications

• Vaccine development
• Viral response
• Auto-immune disorders
• Treatment selection
• Cancer response
• ...

AbSeq

Most NEBNext Products

e.g.

• Ultra II RNA  
• Ultra II DNA
• Small RNA

standard adapter sequences, open source analysis methods

Innovative Enzymes,

Standard Analysis

AbSeq

Unique Method,

Complex Analysis

Customers may not be sequencing experts

Others in this space bundle analysis as a service

Abseq Workflow

300-500 bp

Data Analysis Challenges

• Need to stitch reads to span variable regions (2x300)
• Cannot align to reference DB, need to discover new
• Significant amplification -> need UMIs
• Customers should see details of analysis

pRESTO + Galaxy to the Rescue

Analysis steps

• filter for full length reads
• identify primers and UMI
• build consensus from UMI clusters
• stitch paired reads for single "long" read
• tag C regions (identify isoforms)
• generate QC report
• MigMAP for annotation

User interface

Example output: Reads

Example output: Isoforms

Example output: Clean seqs

@CTGCTTATGATAAAGAT|PRCONS=TS-shift2|SEQORIENT=F|CREGION=Human-TRA InternalC|CONSCOUNT=26568|DUPCOUNT=146
TGGATATTTGGGATGACTTGGAGCTTTGTTCCAGTTCCA...TTGTTTTGGTC
+
{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{{...{{{{{{{{{{{