TITLE: spotPCR: A Rapid and Efficient Approach for Indexing Individual Template Molecules using Unique Molecular Identifiers
AUTHORS: Jason D Limberis

[DESCRIPTION]
Low-frequency mutations provide valuable insights in various fields, including drug resistance identification, cancer and infectious disease research. One promising strategy to enhance the sensitivity and specificity of mutation detection is the incorporation of unique molecular identifiers (UMIs) during polymerase chain reaction (PCR) amplification and before deep sequencing. However, conventional methods for UMI incorporation often necessitate multiple labor-intensive steps. spotPCR (Specific Primer Limited Unique Molecular Identifier Tagging PCR) overcomes these challenges, streamlining the UMI tagging process.

[STEPS]
SECTION: Stage 1 PCR
1. 
   AB1COMPONENTVolume (µl)25X Q5 Reaction Buffer1035X Q5 High GC Buffer10410 mM dNTPs15Q5 High-Fidelity DNA Polymerase0.5610µM Forward primer 11710µM Reverse primer 21820 mg/ml BSA59Template DNA (~1ng/ul)510Nuclease-Free Water18.5
 
     ABCD1StepTemp (C)Time (s)Cycles2Denaturation9812013Denaturation9810104Annealing64155Extension72456Extension4Forever1

SECTION: Stage 2  PCR
2. Add the below into the reaction while at 4 °C and proceed to the second PCR cycling 
   AB1COMPONENTVolume (µl)210µM Forward primer 20.15310µM Reverse primer 10.15410µM Forward Illumina adapter primer0.35510µM Reverse Illumina adapter primer0.35

 
     ABCD1StepTemp (C)Time (s)Cycles2Denaturation9812013Denaturation9810104Annealing64155Extension72456Extension4Forever1

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: 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)
SECTION: Analysis
21. Link to script (click here)
./spotSeq_process.sh \
  -R1 <Read1.fastq> \
  -R2 <Read2.fastq> \
  -Ref_name <Reference.fasta> \
  -sample_name <SampleName> \
  -threads <Threads> \
  -umi <UMI_Barcode_Pattern>

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