TITLE: hissPCR: A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay.
AUTHORS: Jason D Limberis, Alina Nalyvayko, Joel Ernst, john.metcalfe

[DESCRIPTION]
Targeted amplicon sequencing to identify pathogens, resistance-conferring mutations, and strain types is an important tool in diagnosing and treating infections. However, due to the short read limitations of Illumina sequencing, many applications require the splitting of limited clinical samples between two reactions. Here, we outline hairpin Illumina single-tube sequencing PCR (hissPCR) which allows for the generation of overlapping amplicons containing Illumina indexes and adapters in a single tube, effectively extending the Illumina read length while maintaining reagent and sample input requirements.

[STEPS]
SECTION: Stage 1 PCR
2. 
      COMPONENT  FINAL C.  50ul    5X Reaction Buffer  1X  10    5X GC Buffer  1X  10    10 mM dNTPs  200   µM  1    AtpE_stilPCRm1_F1  0.1µM  0.5    AtpE_stilPCRm1_R1  0.1µM  0.5    Rv0678_stilPCRm1_F1  0.1µM  0.5    Rv0678_stilPCRm1_R2  0.1µM  0.5    Template DNA  –  2    Q5 High-Fidelity DNA Polymerase  0.02   U/µl  0.5    Nuclease-Free Water  –  19.5  


 

 As soon as the sample hits 4 °C, proceed to step 2. 
SECTION: Stage 2 PCR
3. Add the below into the reaction while at 4 °C and proceed to the second PCR cycling
      COMPONENT  FINAL C.  50ul    5X Reaction Buffer  1X  10    5X GC Buffer  1X  10    10 mM dNTPs  200   µM  1    AtpE_stilPCRm1_F1  0.1µM  0.5    AtpE_stilPCRm1_R1  0.1µM  0.5    Rv0678_stilPCRm1_F1  0.1µM  0.5    Rv0678_stilPCRm1_R2  0.1µM  0.5    Template DNA  –  2    Q5 High-Fidelity DNA Polymerase  0.02   U/µl  0.5    Nuclease-Free Water  –  19.5  
 
 
 


SECTION: Bead cleanup
4. Add 40 µL of resuspended AMPure XP beads to each reaction in a PCR tube
Mix by pipetting 10x
Incubate 5 min at 65 Room temperature
Place on magnet
Wash 2x with 200 µL freshly-prepared 70 % (v/v) ethanol
Air dry for30 s, don't allow the beads to become cracked
Remove the tubes from the magnetic rack
Add 50 µL 10 mM Tris-HCl pH 8.0 with 50 mM NaCl
NOTE: The BSA in the reaction causes the beads to clump.
Flick the tubes to partially resuspend the beads
Mix by pipetting 10x
Incubate 5 min at 65 Room temperature
Place on the magnet, aspirate  50 µL of the eluant into a new tube
Run  10 µL on a 0.8% agarose gel

SECTION: Optional: Determine the proportion of amplicons with both Illumina adapters via qPCR
5. Library quantification can be done using a commercial kit such as the KAPA Library Quantification Kits or using the below custom protocol.

Dilute the cleaned up amplicons 1:100.
Make the master mix below using Illumina_quant_Inner primer set and a second master mix for Illumina_quant_Outer primer set.
      COMPONENT  FINAL C.  50ul    5X Reaction Buffer  1X  10    5X GC Buffer  1X  10    10 mM dNTPs  200   µM  1    AtpE_stilPCRm1_F1  0.1µM  0.5    AtpE_stilPCRm1_R1  0.1µM  0.5    Rv0678_stilPCRm1_F1  0.1µM  0.5    Rv0678_stilPCRm1_R2  0.1µM  0.5    Template DNA  –  2    Q5 High-Fidelity DNA Polymerase  0.02   U/µl  0.5    Nuclease-Free Water  –  19.5  
Aliquot 8 µL to each well and add in 2 µL of the diluted amplicon.
SECTION: Optional: Determine the proportion of amplicons with both Illumina adapters via qPCR
6.  
 

SECTION: Optional: Determine the proportion of amplicons with both Illumina adapters via qPCR
7. The ratio of the Illumina _quant_Inner primer set to the Illumina _quant_Outer primer set will give the proportion of the DNA in the tube that contain both Illumina adapters and is sequencable (use this for pooling). While the melt curve will provide information on the proportion of each amplicon in the sample.
SECTION: Expected Result
8.     
SECTION: Primer design
1. Primers must be designed as shown in the figure below, if any amplicon is larger than the 2 x sequencing length, the inner portion of the amplicon will not be covered.  We have developed a pipeline to assist in creating primers, which is available here: hisPCR primer design, and is used as follows. 
  
 


SECTION: Depth and Pooling Calculations
9. The amount of amplicon to be pooled and loaded can be calculated using the Illumina Sequencing Coverage Calculator by selecting DNA input then custom content and
depends on numerous factors, including the amplicon size and number, and the kit and device used.  An example output is shown below.
 
      Application or   product:  Custom Content    Genome or region size (Mbases)  0.001    Read length  300    On target (%)  100    Coverage (x)  50000    Duplicates (%)  0      Instrument  MiSeq    Run type  v3  Reagents    Clusters  25,000,000  per flow cell    Output per unit (flow cell or lane)  7,500,000,000  per flow cell    Exceeds maximum read length?  Does not exceed maximum (2x300)    Number of units per sample (flow cell or   lane)  0.007 flow cells    Samples per unit (flow cell or lane)  150/flow cell Most Illumina library prep kits enable up to 96 indexes;   Nextera XT up to 384 indexes.    Comments  Upgraded software; MCS v2.3 or later; MiSeq Reagent Kit v3 (150/600)    Product  MiSeq Reagent Kit v3    Link  http://www.illumina.com/products/miseq-reagent-kit-v3.html  
 
SECTION: Data Analysis
10. The sequence data can be performed using most amplicon processing pipelines; however, the primer sequences must be trimmed from the reads to eliminate their effect on mutation identification. We have developed a pipeline to process data which is available at hissPCR analysis with further details, below is its basic usage command.