TITLE: Prospecting for zoonotic pathogens using targeted DNA enrichment
AUTHORS: Egie Enabulele

[DESCRIPTION]
There are over 60 zoonoses linked to small mammals, including some of the most devastating pathogens in human history. Meanwhile, millions of museum-archived tissues are available to understand natural history of these pathogens. Our goal is to maximize the value of museum collections for pathogen-based research using targeted sequence capture. To this end, we have generated a probe panel that includes 39,916, 80bp RNA probes targeting 32 pathogen groups, including bacteria, helminths, fungi, and protozoans. Laboratory generated, mock control samples show that we are capable of enriching targeted loci from pathogen DNA 2,882 to 6746-fold. Further, we were able to identify bacterial species inmuseum-archived samples, including Bartonella, a known human zoonosis. These results show that probe-based enrichment of pathogens is a highly customizable and efficient method for identifying pathogens from museum-archived tissues.

[GUIDELINES]
Aseptic Protocols
Wear disposable gown over lab coat during DNA extraction process.
Clean work surfaces with 3% sodium hypochlorite solution.
Aliquot all reagents under a Biological Safety Cabinet.
Single use of reagents aliquot on each work day.
Space out samples by leaving one empty rack space between Microcentrifuge tubes.
Process a sample at a time and sterilize gloves with 70% ethanol between samples.

[STEPS]
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
1. Put ~ 10 mg – 20 mg of tissue in 1.5 mL microcentrifuge tubes (Note: for lysed tissue, put 180 µL of lysate in 1.5 mL microcentrifuge tubes and proceed to step iii)
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
2. Add 180 µL of ATL buffer
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
3. Add 20 µL of Proteinase K
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
4. Vortex at 2000 rpm for 10 sec. and spin down in microcentrifuges for 10 sec.
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
5. Put sample in Dry Bath preheated at 56°C overnight
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
6. Vortex at 2000 rpm for 10 sec.
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
7. Add 200 µL of Buffer AL
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
8. Add 200 µL of Absolute ethanol
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
9. Vortex at 2000 rpm for 3 min
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
10. Pipet the mixture into a DNeasy Mini spin column placed in a 2 mL collection tube.
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
11. Centrifuge at 8000 rpm for 1 min. Discard the flow-through and collection tube.
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
12. Place the spin column in a new 2 mL collection tube. Add 500 μL Buffer AW1.
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
13. Centrifuge at 8000 rpm for 1 min. Discard the flow-through and collection tube.
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
14. Place the spin column in a new 2 mL collection tube, add 500 μL Buffer AW2
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
15. Centrifuge at 14,000 rpm for 3 min. Discard the flow-through and collection tube
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
16. Transfer the spin column to a new 1.5 mL microcentrifuge tube.
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
17. Elute the DNA from the spin column by adding 50 μL of 10 mM Tris-HCl, pH 8.0 (pre heated at 56°C for 10 min.) 
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
18. Incubate for 5 min at room temperature
SECTION: DNA extraction protocol (slight modification from DNeasy® Blood and Tissue Kit protocol for tissues)
19. Centrifuge at 8000 rpm for 1 min, discard spin column and quantify DNA with Qubit Fluorimeter using the Quant-iT™ dsDNA Broad Range Assay.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
20. Put 500 ng DNA per sample in 0.65 mL PCR tubes
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
21. Add enzymatic fragmentation reagents (KAPA Hyperplus protocol)
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
22. Mix by pipetting and spin down for 5 sec
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
23. Incubate at 37°C for 10 min in thermocycler (KAPA Hyperplus protocol)
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
24. Add End-repair and A tailing reagents (KAPA Hyperplus protocol)
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
25. Mix by pipetting and spin down for 5 sec.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
26. Incubate at 65°C for 30 min in thermocycler (KAPA Hyperplus protocol)
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
27. Add Adapter ligation reagents (KAPA Hyperplus protocol)) Note: IDT xGen Stubby Adapter (from the IDT xGen Stubby Adapter-UDI Primers kit) is added to the reaction (replacing the KAPA Adapter stock) to make the required reaction volume.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
28. Mix thoroughly by pipetting and spin down for 10 sec.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
29. Incubate in at 20°C for an hour in thermocycler (Note: the lid temperature should be turned off)
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
30. Post ligation cleanup (KAPA Hyperplus protocol). Note: mix beads and libraries by pipetting.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
31. Elute DNA library in 25 µL 10 mM Tris-HCl, pH 8.0
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
32. Add 20 µL of ligated library in 0.65 mL PCR tubes 
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
33. Add Library Amplification reagents (i.e. KAPA HiFi HotStart ReadyMix (2X) from the KAPA Hyperplus kit) Note: IDT xGen UDI Primers (from the IDT xGen Stubby Adapter-UDI Primers kit) replaces the KAPA Library Amplification Primer Mix (10X) in the reaction.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
34. Mix thoroughly by pipetting and spin down for 10 sec
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
35. Place tubes in thermocycler for 4 cycles of library PCR amplification (KAPA Hyperplus protocol).
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
36. Post library amplification cleanup (KAPA Hyperplus protocol). Note: mix beads and libraries by pipetting.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
37. Elute amplified DNA library in 53 µL 10 mM Tris-HCl, pH 8.0. Note: use 50 µL of amplified DNA library for first DNA library size selection.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
38. Perform first DNA library size selection (KAPA Hyperplus protocol) by mixing 50 µL of amplified DNA library and 25 µL of KAPA Pure Beads. Note: mix beads and libraries by pipetting.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
39. Perform second DNA library size selection (KAPA Hyperplus protocol) by mixing 70 µL of first size cut DNA library and 10 µL of KAPA Pure Beads. Note: mix beads and libraries by pipetting.
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
40. Elute second size cut DNA library in 22 µL PCR grade water (recover ~20 µL of final DNA library)
SECTION: NGS libraries preparations (slight modification from KAPA Hyperplus kit and combined with IDT xGen DNA Library Prep. EZ kit protocols)
41. Use 1 µL of final DNA library for quality control base pairs sizes and concentration estimation (Agilent Technologies D1000 Screen Tape protocol and Agilent 4200 TapeStation®)
SECTION: DNA Target Capture/Hybridization (slight modification from the high sensitivity protocol of myBaits® v.5 (Daicel Arbor Biosciences) Hybridization capture for NGS.
42. Pool between 4 to 16 DNA libraries with similar DNA concentrations together into 1.5 mL tube
SECTION: DNA Target Capture/Hybridization (slight modification from the high sensitivity protocol of myBaits® v.5 (Daicel Arbor Biosciences) Hybridization capture for NGS.
43. Concentrate the pool of samples to 7 µL with a speedvac vacuum concentrator (Note: if volume drops below 7 µL, make up the difference by adding the appropriate volume of PCR grade water)
SECTION: DNA Target Capture/Hybridization (slight modification from the high sensitivity protocol of myBaits® v.5 (Daicel Arbor Biosciences) Hybridization capture for NGS.
44. First round of enrichment at 63˚C for 18 hours (myBaits® v.5 protocol)
SECTION: DNA Target Capture/Hybridization (slight modification from the high sensitivity protocol of myBaits® v.5 (Daicel Arbor Biosciences) Hybridization capture for NGS.
45. 15 cycles of enriched libraries PCR amplification (myBaits® v.5 protocol)
SECTION: DNA Target Capture/Hybridization (slight modification from the high sensitivity protocol of myBaits® v.5 (Daicel Arbor Biosciences) Hybridization capture for NGS.
46. Second round of enrichment at 65˚C for 18 hours (myBaits® v.5 protocol)
SECTION: DNA Target Capture/Hybridization (slight modification from the high sensitivity protocol of myBaits® v.5 (Daicel Arbor Biosciences) Hybridization capture for NGS.
47. 15 cycles of enriched libraries PCR amplification (myBaits® v.5 protocol)
SECTION: DNA Target Capture/Hybridization (slight modification from the high sensitivity protocol of myBaits® v.5 (Daicel Arbor Biosciences) Hybridization capture for NGS.
48. Use 1 µL of enriched library for quality control base pairs sizes and concentration estimation (Agilent Technologies D1000 Screen Tape protocol and Agilent 4200 Tapestation®).
SECTION: DNA Target Capture/Hybridization (slight modification from the high sensitivity protocol of myBaits® v.5 (Daicel Arbor Biosciences) Hybridization capture for NGS.
49. Enriched libraries quantification (KAPA Library Quantification Kit/protocol).
SECTION: DNA Target Capture/Hybridization (slight modification from the high sensitivity protocol of myBaits® v.5 (Daicel Arbor Biosciences) Hybridization capture for NGS.
50. Enriched libraries combined into an equimolar pool for subsequent Illumina Hi-Seq 2500 sequencing.