TITLE: stilPCR (single-tube Illumina long read PCR) increases the effective sequencing length of Illumina, targeted next-generation sequencing
AUTHORS: Jason D Limberis, Roland Nagel, Soumitesh Chakravorty, Dena Block, Scott Dewell, Alina Nalyvayko, Zach Howard, Grant Theron, Rouxjeane Venter, John Metcalfe

[DESCRIPTION]
Identifying pathogens, resistance-conferring mutations, and strain types through targeted amplicon sequencing is an important tool. However, due to the limitations of short read sequencing, many applications require the division of limited clinical samples. Here, we present stilPCR (single-tube Illumina long read PCR), which allows the generation of hemi-nested amplicons in a single tube, with Illumina indexes and adapters, effectively increasing the Illumina read length without increasing the input requirements of reagents or sample. We have successfully utilized stilPCR on clinical sputum from tuberculosis patients to detect drug resistance mutations.

[STEPS]
SECTION: Stage 1 PCR
1.  
      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
2. Add in the below to the same reaction tube.
      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
3. Add 25 µL (1X) of resuspended AMPure XP Beads to the sample
Mix by pipetting 10x
SECTION: Bead cleanup
4. Incubate 2 min at 65 Room temperature
SECTION: Bead cleanup
5. Place on the magnet, allow the beads to aggregate, and remove and discard the supernatant
SECTION: Bead cleanup
6. Add 200 µL 70 % (v/v) ethanol and incubate (still on the magnet) for 30 s
SECTION: Bead cleanup
6.1. Remove the supernatant
SECTION: Bead cleanup
6.2. Repeat   for a total of 2 washes
SECTION: Bead cleanup
7. Air dry for30 s, don't allow the beads to become cracked
SECTION: Bead cleanup
8. Immediately after the bead pellet becomes opaque, remove the tube from magnetic rack and resuspend in 20 µL of Low EDTA Tris Buffer. Ensure all beads are in solution.

SECTION: Bead cleanup
9. Incubate at room temperature for 5 min

SECTION: Bead cleanup
10. Place on magnetic rack, wait for the solution to become clear ~2 min, and transfer the eluted DNA to a new well-labeled tube

SECTION: Optional: NEB Illumina Quantification
11. Thaw the NEBNext Library Quant Master Mix and NEBNext Library Quant Primer Mix. Ensure mixing of NEBNext Library Quant Primer Mix by vortexing. Place reagents on ice.
SECTION: Optional: NEB Illumina Quantification
12. Thaw the NEBNext Library Quant DNA Standards, tubes 1–6. 
Mix by pulse vortexing on a low setting. Briefly spin to collect material from the sides of the tubes. Place on ice.
SECTION: Optional: NEB Illumina Quantification
13. Thaw the NEBNext Library Quant Dilution Buffer (10X). Mix well by vortexing. 
Centrifuge briefly to collect material from the sides of the tube. 
Place on ice.
SECTION: Optional: NEB Illumina Quantification
14. Add 100 µL NEBNext Library Quant Primer Mix to the tube of NEBNext Library Quant Master Mix (1.5 mL). Mix by vortexing. Write the date on the master mix tube to indicate that primer mix has been added.

SECTION: Optional: NEB Illumina Quantification
15. Dilute the NEBNext Library Quant Dilution Buffer (10X) 1:10 with nuclease-free water. Mix by vortexing. 
Prepare sufficient buffer for quantitating the desired number of libraries, allowing 1.2 mL for each library.

SECTION: Optional: NEB Illumina Quantification
16. Prepare a 1:1,000 dilution of each library sample in NEBNext Library Quant Dilution Buffer (1X)
SECTION: Optional: NEB Illumina Quantification
17. Aliquot 16 µL NEBNext Library Quant Master Mix (with primers) to each well

SECTION: Optional: NEB Illumina Quantification
18. Add-in 4 µL of sample or standard per well

SECTION: Optional: NEB Illumina Quantification
19.  
     ABCD1StepTemp (C)Time (s)Cycles2Denaturation9812013Denaturation9810104Annealing64155Extension72456Extension4Forever1

A denaturation/melt curve can be included if desired, but is optional.
 
      Sample  Conc. (pM)    DNA Standard 1  100    DNA Standard 2  10    DNA Standard 3  1    DNA Standard 4  0.1    DNA Standard 5  0.01    DNA Standard 6  0.001  
 
SECTION: Optional: NEB Illumina Quantification
20. Adjusted Conc. = Calculated Conc. × 399 / library size (bp)

For library size, use the average of the two amplicon sizes.

SECTION: Data processing
21. Data processing can be done using the stilPCR pipelineavailable here (link). 


 

SECTION: Expected Outcome
22. 

 

Successful sequencing of rv0678 and its promoter region using stilPCR. The plot shows the coverage (y-axis) at each position (x-axis) along the sequence amplicons. The coding region is denoted by the grey line at the top of the figure. The positions of forward primer sequencing amplicons are shown in orange at the bottom of each plot, with reverse primer amplicons in pink.