TITLE: iSeq Bacterial WGS Protocol using a quarter of the reagents
AUTHORS: Sonsiray Alvarez Narvaez, Zhenyu Shen, Lifang Yan

[DESCRIPTION]
This is an adaptation of protocol https://dx.doi.org/10.17504/protocols.io.bij8kcrw in which initial the initial DNA concentration and library prep reagent volumes have been reduced to a quarter.

(The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.)

[GUIDELINES]
Wear gloves and change gloves often to avoid cross contamination. 
Consider using separate laboratory coats and disposable sleeve covers for bacterial propagation, DNA extraction, and library preparation.
Be sure to use clean, aerosol resistant pipette tips for addition of reagents to samples and in between samples to avoid cross-contamination.

[STEPS]
SECTION: Bacterial propagation and DNA extraction
1.  See "guidelines and warnings" for general precautions. 

Assemble the following materials and reagents:

     Kit and   Reagent      Components       Amount for 1 sample       Initials        Qiagen DNeasy Blood &   Tissue Kit       Buffer ATL      180 μL               Proteinase K      20 μL               Buffer AL      200 μL               Buffer AW1 (98mL   concentrate+130mL 200 proof ethanol)      500 μL               Buffer AW2(66mL   concentrate+160mL 200 proof ethanol)      500 μL               DNeasy Mini spin column +   additional collection tubes      NA               Qiagen #19086      Buffer EB (for elution of samples)      100 μL               Qiagen #19101      RNase A      4 μL               Enzymatic   lysis (ELB)-lysozyme mixture for Gram positive isolates only      180 μL ELB and 3.6 mg   lysozyme  (ELB:   5mL 1 M Tris-HCl, pH 8.0, 1 mL 0.5M EDTA, 3 mL Triton X-100, add   molecular-grade water  to bring the   volume up to 250 mL, store at RT for 1 year)      180 μL               Lysozyme-   for Gram positive isolates only      (for making ELB)      NA               Triton   X-100- for Gram positive isolates only      (for making ELB)      NA               1 M   Tris-HCl, pH 8.0- for Gram positive isolates only      (for making ELB)      NA               200 proof or 96-100%   ethanol, Molecular biology grade      NA      200 μL               Water, Molecular biology   Grade      For resuspending colonies,   preparing 80% EtOH, and dilution of DNA      200 μL               Inoculating   loop (1 μL) (BD, #220215)      NA      NA          
 
SECTION: Bacterial propagation and DNA extraction
2. Frozen isolates should be sub-cultured on Trypticase Soy + 5% Sheep Blood Agar plates (BAPs) or equivalent media. If the culture appears pure, pick an isolated colony, and streak it on a fresh BAP; incubate at 35°C overnight (24±2 hours) in aerobic conditions.
SECTION: Bacterial propagation and DNA extraction
3. DNA extraction may be performed either from a colony from the second BAP or from an overnight liquid culture pellet from that colony.

From broth: Transfer 1-1.5 mL of culture to a labelled microcentrifuge tube and centrifuge at 5,000 g for 10 minutes. Remove the supernatant and store the pellet in the tube at -20°C until DNA is extracted.

Direct from colony: (when ready to proceed directly with extraction) from bacterial colonies on plate, fill out a 1 μL loop with overnight growth and add to a labelled microcentrifuge tube containing the appropriate extraction buffer for gram positive or gram negative bacteria.

For Gram Positive bacteria, proceed to step 4. 

For Gram Negative bacteria, proceed to step 9.
SECTION: Bacterial propagation and DNA extraction
4. Gram Postive Bacteria.

From broth: Add 180 μL of prepared enzymatic lysis (ELB)-lysozyme mixture to the tube and resuspend the pellet. Proceed to step 5.

From colony: Add overnight growth directly to 180 μL of prepared enzymatic lysis (ELB)-lysozyme mixture to the tube and resuspend the pellet. Proceed to step 5.
SECTION: Bacterial propagation and DNA extraction
5. Incubate at 56°C for 30 minutes, vortexing periodically.
SECTION: Bacterial propagation and DNA extraction
6. Optional: Add 4 μL of RNase A to each tube, vortex and then incubate at RT for 3-5 minutes.
SECTION: Bacterial propagation and DNA extraction
7. Add 25 μL Proteinase K and 200 μL Buffer AL to the tube.
SECTION: Bacterial propagation and DNA extraction
8. Mix thoroughly by vortexing, and incubate at 56°C for 30 minutes. Proceed to step 14.
SECTION: Bacterial propagation and DNA extraction
9. Gram Negative Bacteria.

If the ATL buffer solution is cloudy and has precipitate formation, be sure to heat the solution at 50 - 55°C until solution is clear.

From broth: Add 180 μL Buffer ATL to the tube and resuspend the pellet. Add 20 μL Proteinase K to the tube, then vortex. Proceed to step 10.

From colony: Add overnight growth directly to 180 μL Buffer ATL to the tube and resuspend the pellet. Add 20 μL Buffer proteinase K to the tube, then vortex. Proceed to step 10.
SECTION: Bacterial propagation and DNA extraction
10. Incubate at 56°C for a minimum of 1 hour or up to overnight, vortexing periodically.
SECTION: Bacterial propagation and DNA extraction
11. Optional: Add 4 μL of RNase A to each tube, vortex and then incubate at RT for 3-5 minutes.
SECTION: Bacterial propagation and DNA extraction
12. Add 200 μL Buffer AL to the tube.
SECTION: Bacterial propagation and DNA extraction
13. Mix thoroughly by vortexing, and incubate at 56°C for 10 minutes. Proceed to step 14.
SECTION: Bacterial propagation and DNA extraction
14. Add 200 μL ethanol (200 proof) to each tube and mix thoroughly by vortexing followed by a brief pulse spin.
SECTION: Bacterial propagation and DNA extraction
15. Pipet the mixture from step 14 into a labelled DNeasy Mini spin column. Centrifuge at 6,000xg for 1 minute. Discard flow-through and the collection tube.
SECTION: Bacterial propagation and DNA extraction
16. Add 500 μL Buffer AW1, and centrifuge for 1 minute at 6,000xg (8,000rpm). Discard the flow-through and the collection tube.
SECTION: Bacterial propagation and DNA extraction
17. Add 500 μL Buffer AW2, and centrifuge for 1 minute at 6,000xg (8,000rpm). Discard the flow-through and the collection tube.
SECTION: Bacterial propagation and DNA extraction
18. Transfer the spin column to a new collection tube.
SECTION: Bacterial propagation and DNA extraction
19. Centrifuge for 1 minute at 18,000-20,000xg (14,000rpm) to dry.
SECTION: Bacterial propagation and DNA extraction
20. Place the spin column in a clean, labelled 1.5 mL microcentrifuge tube, and pipet 100 μL Buffer EB directly onto the DNeasy membrane.
SECTION: Bacterial propagation and DNA extraction
21. Incubate at room temperature for 1 minute, and then centrifuge for 1 minute at 18,000-20,000xg (14,000rpm) to elute. Record which speed used.
SECTION: Bacterial propagation and DNA extraction
22. Discard spin column and store DNA sample at 4°C for up to 48 hours. For long term storage (>48 hours), store DNA sample at -20°C.
SECTION: 2.	DNA quantification and quality check
23. DNA quantification using the Qubit. 

The following protocol is for 4 samples. If needed, scale the volumes down or up depending on the sample number. The DNA input necessary for this procedure is >100ng. However, an input larger than 500ng is not recommended. Therefore, quantification is a recommended step to ensure proper amount of DNA is added.
SECTION: 2.	DNA quantification and quality check
24. Download the   and fill in the isolate identifiers in column B. The blue boxes in the workbook should be filled in while following this protocol. 

SECTION: 2.	DNA quantification and quality check
25.  Assemble the following materials and reagents:
     DNA quantification using Qubit Fluorometer        Equipment, Kit, and Reagent      Components      Amount per sample      Amount for 4 samples + 2 standards and +1 extra           Qubit 2.0, 3.0, or 4.0 Fluorometer  Invitrogen, #: Q32866      NA      NA      NA           Qubit dsDNA BR Assay Kit   (2-1000ng), #: Q32850, 1store at room temperature  2store protected from light  3store at 4°C      Qubit   dsDNA BR Buffer1      196 μL      1372   μL           Qubit dsDNA BR Reagent   (200X)1,2       1 μL      7 μL           Qubit dsDNA BR Standard #13      10 μL      10 μL           Qubit dsDNA BR Standard #23      10 μL      10 μL           Qubit dsDNA HS Assay Kit   (0.2-100ng) #: Q32854,   1store at room temperature  2store protected from light  3store at 4°C      Qubit   dsDNA HS Buffer1      196 μL      1372   μL           Qubit dsDNA HS Reagent   (200X)1,2       1 μL      7 μL           Qubit dsDNA HS Standard #13      10 μL      10 μL           Qubit dsDNA HS Standard #23      10 μL      10 μL           Qubit assay tubes, #:   Q32856      NA      NA      NA           Water, Molecular biology   Grade       For DNA dilution      NA      NA      
 
SECTION: 2.	DNA quantification and quality check
26. Wear gloves to keep sides of reagent tubes clean and to protect yourself from the reagents. Remove components of Qubit dsDNA BR Assay Kit stored at 4°C and ensure they are at room temperature before use. Be sure to vortex and pulse spin the standards and reagent dye.
SECTION: 2.	DNA quantification and quality check
27. Prepare the Qubit working solution by diluting 7 μL Qubit dsDNA BR Reagent 200X in 1393 μL Qubit dsDNA BR Buffer in a clean, low-bind plastic tube.
SECTION: 2.	DNA quantification and quality check
28. Label the tops of Qubit assay tubes to correspond to sample label and BR dsDNA standard label. Do not label the tube sides.
SECTION: 2.	DNA quantification and quality check
29. Add 190 μL of Qubit working solution to each of the Qubit assay tubes used for Standard #1 and #2. 
SECTION: 2.	DNA quantification and quality check
30. Add 196 μL Qubit working solution to individual Qubit assay tubes used for each sample. 
SECTION: 2.	DNA quantification and quality check
31. Add 10 μL of each Qubit Standard to the appropriate standard tube, and add 4 μL DNA sample to sample tubes, then mix by vortexing. Briefly pulse spin to assure there are no bubbles present. Note: the input range for the Qubit platform is 1-20 μL, and the volume can be adjusted if desired (as long as the final volume is 200 μL).
SECTION: 2.	DNA quantification and quality check
32. Allow all tubes to incubate at room temperature for 2 minutes. Avoid unnecessary handling of tubes as transfer of heat from your hands may lower the reading.
SECTION: 2.	DNA quantification and quality check
33. On the Home screen of the Qubit 2.0/3.0/4.0 Fluorometer, press DNA, then select dsDNA Broad Range as the assay type. The “Standards” screen is displayed.
SECTION: 2.	DNA quantification and quality check
34. On the Standards screen, press Yes to read the standards.
SECTION: 2.	DNA quantification and quality check
35. Insert the tube containing Standard #1 into the sample chamber, close the lid, then press Read. When the reading is complete, remove Standard #1.
SECTION: 2.	DNA quantification and quality check
36. Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read. When the reading is complete, remove Standard #2.
SECTION: 2.	DNA quantification and quality check
37. Select 4 µL for Volume of Sample and insert a sample tube into the sample chamber, close the lid, then press Read. When the reading is complete, record the calculated stock concentration. Then remove the sample tube.
SECTION: 2.	DNA quantification and quality check
38. Repeat reading until all samples have been read.
SECTION: 2.	DNA quantification and quality check
39. Record the Qubit readings in the Initial Dilution tab, Column I, DNA Concentration, of the DNA Prep workbook.

SECTION: 2.	DNA quantification and quality check
40. DNA quantification and/or quality check using NanoDrop.

     Equipment, Kit, and Reagent      Components      Amount per sample      Amount for 4 samples + 2 standards and +1 extra      Initials        NanoDrop (ThermalFisher)      NA      NA      NA          
 
SECTION: 2.	DNA quantification and quality check
41. Select Nucleic Acid assay on computer or touchscreen interface of NanoDrop. You may need to do further selection to find the assay specifically for dsDNA.
SECTION: 2.	DNA quantification and quality check
42. Clean the upper and lower pedestals of the sample loading area with a laboratory wipe.
SECTION: 2.	DNA quantification and quality check
43. Select Nucleic Acid assay on computer or touchscreen interface of NanoDrop. You may need to do further selection to find the assay specifically for dsDNA.
SECTION: 2.	DNA quantification and quality check
44. Load 2 μL water on the lower pedestal and close the upper arm gently. Click “Initiate”. Wipe off water on the lower and upper pedestals.
SECTION: 2.	DNA quantification and quality check
45. Load 2 μL EB on the lower pedestal and close the upper arm gently. Click “Blank”. Wipe off water on the lower and upper pedestals.
SECTION: 2.	DNA quantification and quality check
46. Load 2 μL of sample on the lower pedestal and close the upper arm gently. Click “Measure”. Record concentration, 260/280, and 260/230. When recording concentration, make sure dsDNA assay is used. Wipe off water on the lower and upper pedestals. Load the next sample for test. It is not necessary to clean with water between sample tests.
SECTION: 2.	DNA quantification and quality check
47. Record the NanoDrop concentration readings in the Initial Dilution tab, Column H, DNA Concentration, of the DNAFlex workbook.
SECTION: 2.	DNA quantification and quality check
48. Record the 260/280 and 260/230 ratios in Columns F and G, Initial Dilution tab, of the DNA Flex Workbook. Refer to the Illumina DNA Prep guide for recommended ratios.
SECTION: 2.	DNA quantification and quality check
49. Clean both upper and lower pedestals of sample loading area with water and laboratory wipes. Close the upper arm.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
50.  Assemble the following materials and reagents:
     Kit and Reagent      Components      Amount for each sample        Magnet stand, to handle   tubes or plates, depending upon sample throughput      NA      NA        Nextera DNA Flex Library   Prep Kit, Box 1      SPB      45 μL   + 15 μL        TSB      10 μL        TWB   (Pipette slowly to   minimize foaming.)      100 μL   X 3        Nextera DNA Flex Library   Prep Kit, Box 2      TB1      10 μL        EPM      20 μL        RSB      32 μL        Nextera DNA Flex Library   Prep Kit, Box 3       BLT  (Store the BLT stock tube   upright in the refrigerator so that the beads are always submerged in the buffer.   Vortex the stock tube thoroughly until the beads are resuspended. It is not   recommended to centrifuge the stock tube after vortexing.)      10 μL        Ethanol 200 proof   for molecular biology (any supplier)      Fresh 80% ethanol      180 μL X 2        i7 and i5 index  Nextera   DNA CD Indexes (24 indexes, Cat no 20018707)      i7 indices: H705, H706,   H707, H710      5 μL   each        i5 indices: H503, H505,   H506, H517      5 μL   each        PhiX Control v3, 10nM   (Illumina, FC-110-3001)      NA      NA        Ice      NA      NA        DNA      NA      NA   
 
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
51. Preparation of Sample Sheet. 

The steps for Sample Plate and Sample Sheet setup in the Illumina Experiment Manager on the instrument can be completed at any point in this protocol but must be performed prior to the start of the run. However, it is recommended to create the Sample Plate and Sample Sheet at the beginning of library prep. 
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
52. Depending upon desired sample throughput, the samples can be prepped using individual tubes with a magnetic stand device equipped to handle tubes. Alternatively, samples can be prepped using a 96-well format with a magnetic device that handle plates. A 96-well format lends itself for using a multi-channel pipette. 
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
53. Prepare the "Initial dilution" tab from the "DNA Flex Workbook". 

NOTE: The workbook is designed with the following color scheme, in general, the blue colored fields should be filled in.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
54. Confirm or adjust the i7 and i5 indexes listed in Columns D and E. If using indexes purchased in the 96-well plate format, choose positions on the Nextera CD indexes map and note the wells used. 
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
55. Check that the DNA input (ng) is ~75 ng in Column J. If it is below this range, adjust the volume multiplier (change the 2 to a higher number) in the Column J formula so that it falls in this range.  If the original concentration falls above range (DNA is too concentrated), perform a dilution, re-check the concentration, and update row I.

NOTE: We adjusted the concentration of the DNA input to 10ng/μL and we used 7.5 μL.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
56. Add enough nuclease-free water calculated in Column L of the "DNA Flex Workbook" to each sample in a clean PCR tube.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
57. Add volume of DNA adjusted in Column K of the "DNA Flex Workbook" to the sample tubes now containing nuclease-free water.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
58. Mix by pitetting 5-10 times. Close tubes (or seal plate) and store at 4-8º C overnight, or proceed to step 59.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
59. Tagment Genomic DNA.

Remove the SPB, TSB, and TWB from Box 1 and bring to room temperature. The TSB and TWB can then be stored at room temperature. Remove one tube of BLT from Box 3 and bring to room temperature. Thaw the RSB, bring TB1 (from freezer) to room temperature.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
60. Vortex BLT vigorously for 10 seconds, visually check the beads for complete resuspension and repeat as necessary. Vortex the TB1.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
61. Prepare the tagmentation master mix according to the following table:

 

SECTION: 3.	Nextera DNA Flex/Prep Library preparation
62. Vortex the Tagmentation master mix thoroughly to make sure the BLT beads are evenly resuspended in the buffer.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
63. Using fresh tips, transfer 5 µL of Tagmentation master mix to each well containing a sample.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
64. Pipette up and down 10 times mixing the 12.5 µL reaction to resuspend the beads.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
65. Close the PCR tube (or seal the plate with Microseal 'A' or equivalent if using a 96-well format).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
66. Place the PCR tubes into the thermalcycler and run the tagmentation program FLEXTAG. If the thermalcycler does not have the program, create one using the following conditions with a lid pre-heated.

Incubation Conditions: (12.5 µL total reaction)
1.   55°C for 15 minutes
2.   Hold at 10°C

Upon completion of the incubation, remove the PCR tubes from the thermalcycler and proceed to Post Tagmentation Cleanup steps.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
67. Post Tagmentation Cleanup. 

Check for precipitates in the TSB. If present, heat the buffer at 37°C for 10 minutes and vortex until dissolved.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
68. Open the PCR tubes (or remove the seal from plate).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
69. Add 2.5 µL of TSB to the tagmentation reaction.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
70. Gently pipette up and down 10 times, mixing the entire volume to resuspend the beads.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
71. Close the PCR tubes (or seal the plate with Microseal 'A' or equivalent).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
72. Place the PCR tubes into the thermalcycler and run the program FLEXPOST. If the thermalcycler does not have the program, create one using the following with a lid pre-heated.

Incubation Conditions: (now 15 µL total reaction)
1.   37°C for 15 minutes
2.   Hold at 10°C
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
73. Remove the EPM and indices from freezer and allow to come to room temperature while this incubation is running to prepare for later steps.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
74. Remove the PCR tubes from the thermalcycler, quick spin down, then open the tubes (or remove the seal).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
75. Place on the magnet for 3 minutes or until solution is clear.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
76. Use a multichannel pipette, remove the supernatant and discard.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
77. Remove the tubes from the magnet and add 100 µL of TWB directly to the beads. Gently pipette to mix until beads are fully resuspended. 

NOTE: It is challenging to resupend the pellet at this series of washes, check each well to ensure sufficient mixing of beads.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
78. Place the tubes on the magnet for 3 minutes or until solution is clear.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
79. Remove the supernatant and discard.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
80. Repeat steps 77 - 79 one more time for a total of two washes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
81. Remove the tubes from the magnet and add 100 µL of TWB directly to the beads. Gently pipette to mix until beads are fully resuspended.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
82. Place the tubes with TWB on the magnet and allow to incubate until ready to proceed with adding the PCR master mix in the Amplify Tagmented DNA step. The tubes should incubate for at least 3 minutes. It is important to keep the pellet in TWB to prevent over drying of the beads.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
83. Amplify Tagemented DNA. 

Invert the EPM to mix.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
84. Briefly spin the Index tubes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
85. Prepare the PCR master mix according to the following table:

 

SECTION: 3.	Nextera DNA Flex/Prep Library preparation
86. Vortex and spin down the PCR master mix.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
87. Remove the third TWB wash from the samples while on the magnet. Remove any excess liquid from the tubes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
88. Remove the tubes from the magnet and immediately proceed to adding the master mix.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
89. Add 10 µL of the PCR master mix to each sample well. Gently pipette to mix to ensure beads are resuspended.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
90. Open the i7 primer tubes with orange caps and discard the caps. Add 1.25 µL of index primers to the NTA tube. After completion, use orange replacement caps to close the primer tubes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
91. Open the i5 primer tubes with white caps and discard the caps. Add 1.25 µL of index primers to the NTA tube. After completion, use the white replacement caps to close the primer tubes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
92. Use a pipette to mix a minimum of 10 times at a higher volume (30 µL) to ensure throrough mixing. 

NOTE: Be careful to avoid introducing air bubbles.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
93. Close the tubes (or seal the plate with Microseal 'A' or equivalent).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
94. Place the tubes into the thermalcycler and run the following program (FLEXPCR):

PCR Conditions: (12.5 µL total reaction)
1.     68°C for 3 minutes
2.     98°C for 3 minutes
3.     98°C for 45 seconds
4.     62°C for 30 seconds
5.     68°C for 2 minutes
6.     Repeat steps 3-5 an additional 4 times for a total of 5 cycles
7.     68°C for 1 minute
8.     Hold at 10°C
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
95. Remove the tubes from the thermal cycler.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
96. This is a safe stopping point. Store at 4°C for up to 3 days, if you decide to store the tubes (or with plates, replace the Microseal 'A' with a Microseal 'B'). If ready to Clean Up Libraries, proceed to the next step.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
97. Remove the SPB from the refrigerator to equilibrate to room temperature if proceeding to Clean Up Libraries step.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
98. Clean Up Libraries. (The library fragments are free of the BLT at this point).

Before starting, prepare reagents. Dilute fresh 80% ethanol sufficient for all samples:

 



SECTION: 3.	Nextera DNA Flex/Prep Library preparation
99. Be sure the SPB is at room temperature (at least 30 minutes from refrigerator).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
100. Bring RSB to room temperature (from the freezer) and vortex to mix.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
101. Vortex and invert SPB several times to fully re-suspend the beads.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
102. Prepare SPB master mix:

 

NOTE: It is recommended to increase the number of samples 3-4 to ensure sufficient volume of master mix.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
103. If PCR tubes (or plate) were retrieved from cold storage, centrifuge at 280 x g for 1 minute.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
104. Remove the seal.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
105. Place the tubes on the magnet for 3-5 minutes (until supernatant is clear).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
106. Transfer 10 µL of the PCR supernatant (now containing DNA)  to clean tubes (or a different location on the sample plate or into a fresh 96-well plate).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
107. Remove the tubes from magnet and discard.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
108. Vortex and then add 21.25 µL of the SPB master mix prepared in step 102, to each well. Be sure to not carry over extra SPB master mix on the outside of the tips. Wipe the outside of the tips on the reservoir to remove droplets before transferring the SPB master mix to the PCR supernatant.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
109. Pipette to mix a minimum of 10 times until thoroughly mixed (use caution as volume is > 100 µL).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
110. Incubate at room temperature for 5 minutes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
111. Place tubes on the magnet for 5 minutes or until supernatant is clear.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
112. During the incubation, vortex the SPB stock tube thoroughly, and add 3.75 µL to new tubes (one for each sample) (or on the same plate or in a new 96-well plate). Be sure to remove excess SPB from the outside of tips before addition to new tubes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
113. Transfer 31.25 µL of supernatant  to the wells containing the 3.75 µLSPB (stock concentration).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
114. Gently pipette to mix 10 times at a volume of ~85 µL, avoiding the introduction of air bubbles.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
115. Incubate at room temperature for 5 minutes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
116. Place the tubes on the magnet for 5 minutes or until clear.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
117. Remove and discard the supernatant without disrupting the beads.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
118. With the tubes on the magnet, add 180 µL of freshly prepared 80% ethanol and incubate for 30 seconds.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
119. Pipette to remove the ethanol supernatant.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
120. Repeat steps 118 and 119 for a total of two washes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
121. Use a pipette to remove any excess liquid from tubes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
122. Leave beads to air dry on the magnet for 5 minutes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
123. Remove the tubes from the magnet and add 32 µLof RSB to the beads.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
124. Pipette to mix until thoroughly resuspended.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
125. Incubate at room temperature for 2-5 minutes.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
126. Place the tubes onto the magnet for 3 minutes until clear.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
127. Transfer 30 µL of the supernatant into a new tube for each sample (or new well on a 96-well plate for each sample).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
128. If ready, continue to the Pooling Libraries step. Otherwise, this is a safe stopping point. Close the tubes (or seal the plate with a freezer-safe seal) and store at -20°C for up to 30 days.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
129. Pooling of Libraries and dilution of the pool to 1 nM concentration.

Perform quantitation on each of the individual libaries. Qubit HS assay is recommended by Illumina, but a NanoDrop can be used if this is the only instrument available. Enter the results into Column B on the "Normalization and Pooling" tab in the "DNA Flex Workbook". 
 
NOTE:This procedure assumes that all input DNA is >100 ng and therefore would yield a stable library. Nextera DNA Flex library preparation method produces normalized libraries and does not require a dilution-based normalization step.
 
 
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
130. Pool 5 μL per sample into a single 1.5ml tube. Optional: for smaller bacterial genomes, reduce the input volume to 2.5  μL.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
131. Mix by vortexing then spin down.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
132. Quantify the pooled library using the Qubit HS assay or NanoDrop. Record result at the bottom of the “Normalization and Pooling” tab in the “DNA Flex Workbook”. 
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
133. This SOP uses the CFSAN suggested conversion factor of 2.5 which assumes an average fragment size of roughly 600 bp. The “DNA Flex Workbook” will convert the final pooled library input to nM using this conversion factor.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
134. The “DNA Flex Workbook” will then calculate the values necessary to dilute your library to a concentration of 4 nM in a volume of 15 µL (C1V1 = C2V2).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
135. Dilute the final library to 4 nM according to the values on the workbook using RSB.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
136. Add 5 µl of the 4 nM pool to 15 µL RSB to a new low bind microtube to make 1 nM pool.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
137. Vortex briefly, and then quick-spin at 280 x g for 3 sec.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
138. Set aside 1 nM library on ice or this is a safe stopping point. The 1 nM library can be stored at -20ºC for up to 1 month. Stopping at this point will also allow for thawing.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
139. Preparation of Cartridge.

Wear powder-free gloves.Thaw by following one of three ways listed below. Prior to thawing, remove the cartridge from the box but do not open the white foil bag! After thawing, do not refreeze. Store cartridge at -15˚C to -25˚C at least one day before thawing using any of the methods listed below.

 
If cartridge is wet from a water bath, dry with paper towels.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
140. Dilute Pool to 200 pM Loading Concentration for iSeq.

Add 20 µL of 1 nM pool to 80 µl of RSB to a new low bind microtube to produce 100 µL of 200 pM concentration.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
141. Vortex briefly, and then centrifuge at 280 x g for 3 sec.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
142. Spiking of Library with PhiX.

Add 2 µL of 10 nM PhiX to 18 µL of RSB in a new low bind microtube to make 1 nM PhiX. PhiX can be safely stored at -20°C for up to one month at this concentration.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
143. Vortex briefly, and then centrifuge at 280 x g for 3 sec. Set aside on ice.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
144. Further dilute the PhiX in a low bind microtube to 200 pM by adding 20 µL of 1 nM PhiX to 80 µL of RSB.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
145. Vortex briefly, and then centrifuge at 280 x g for 3 sec.
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
146. Add 2 µL of 200 pM PhiX to the tube that contains 100 µL of 200 pM pooled library. (These volumes result in a ~2% PhiX spike-in. Actual percentage varies depending on library quality and quantity).
SECTION: 3.	Nextera DNA Flex/Prep Library preparation
147. Set aside the library with PhiX spike-in on ice. This is the final loading product.
SECTION: iSeq loading and set up
148. Load the Flow Cell.

Avoiding the access window on top of the cartridge, remove the cartridge from the bag. Discard the bag.
SECTION: iSeq loading and set up
149. Invert the cartridge five times to mix reagents. Internal components can rattle during inversion, which is normal.
SECTION: iSeq loading and set up
150. Tap the cartridge (label facing up) on the bench or other hard surface five times to ensure reagent aspiration.
SECTION: iSeq loading and set up
151. Using a new pipette tip, pierce the Library reservoir and push the foil to the edges to enlarge the hole. Discard the pipette tip.
SECTION: iSeq loading and set up
152. Add 20 µL diluted library to the bottom of the reservoir. Avoid touching the foil.
SECTION: iSeq loading and set up
153. Open the white foil package containing the flow cell from the notches. Use within 24 hours of opening.
SECTION: iSeq loading and set up
154. Carefully pull the flow cell out of the package. Touch only the plastic when handling the flow cell. Avoid touching the electrical interface, CMOS sensor, glass, and gaskets on either side of the glass. See the referenced iSeq guide for an illustration.
SECTION: iSeq loading and set up
155. Hold the flow cell by the grip points with the label facing up.
SECTION: iSeq loading and set up
156. Insert the flow cell into the slot on the front of the cartridge. An audible click indicates that the flow cell is in place. When properly loaded, the grip protrudes from the cartridge and the glass is visible from the access window.
SECTION: iSeq loading and set up
157. Set Up a Sequencing Run.

From the control software menu, select Local Run Manager, and then select Open Local Run Manager.
SECTION: iSeq loading and set up
158. Select + Create Run.
SECTION: iSeq loading and set up
159. From the three available options, select Generate FASTQ.
SECTION: iSeq loading and set up
160. Enter Run Name in this format: YYMMDD-Vet-LIRN-LAB [“LAB” is your two-letter state abbreviation listed in Appendix B.] [Example, for a run starting on November 27, 2019 the run name would be entered as 191127-Vet-LIRN-NY].
SECTION: iSeq loading and set up
161. Enter Run Description (e.g. "Practice Samples").
SECTION: iSeq loading and set up
162. Under Run Settings, select the appropriate Library Prep Kit (Nextera DNA CD Indexes-24 index tube.)
SECTION: iSeq loading and set up
163. Index Reads, select the box next to “2.”
SECTION: iSeq loading and set up
164. Read Type, select Paired End.
SECTION: iSeq loading and set up
165. Read Lengths, be sure that 151 has been entered for Read 1.
SECTION: iSeq loading and set up
166. Read Lengths, be sure that 151 has been entered for Read 2.
SECTION: iSeq loading and set up
167. Under Module-Specific Settings, be sure the Adapter Trimming option “On” is selected.
SECTION: iSeq loading and set up
168. For each sample being sequenced, enter the Sample ID, Sample Description, and Sample Project. Do not modify the Sample ID, Sample Description, and Sample Project. It must be entered exactly as given below.

The Sample ID is the same as the Isolate ID listed in Column B of the Initial Dilution Tab of the Flex workbook.

Sample Description is the same as the Organism in Column C of the Initial Dilution Tab of the Flex workbook. If labeling with genus and species a “-“ must be included between the Genus and Species because the sequencer software does not recognize spaces.

For the Vet-LIRN iSeq study: The Sample Project name is: VL-iSeq-LAB, where “LAB” is your two-letter state abbreviation. Do not deviate from this naming format.
SECTION: iSeq loading and set up
169. Under INDEX plate location using the dropdown.
SECTION: iSeq loading and set up
170. Save the run.
SECTION: iSeq loading and set up
171. In the control software, select Sequence from the Home screen. The software opens the door at an angle, ejects the tray, and initiates the series of run setup screens.
SECTION: iSeq loading and set up
172. Load the Cartridge onto the Instrument.

Place the cartridge onto the tray so that the access window faces up and the flow cell is inside the instrument. Do not push the cartridge or tray into the instrument.
SECTION: iSeq loading and set up
173. Select Close Door to retract the cartridge and close the door. A panel appears on the left side of the screen to show information from the scanned consumables. During scanning, you can continue with run setup.
SECTION: iSeq loading and set up
174. Sign in to BaseSpace Sequence Hub.

Sign in to your BaseSpace Sequence Hub by entering your email address and password, and then select Sign In.
SECTION: iSeq loading and set up
175. Under configurations, make sure that “run monitoring and storage” has been checked.
SECTION: iSeq loading and set up
176. Select Run Setup.
SECTION: iSeq loading and set up
177. Select a Run and Review Pre-Run Checks.

Select the run you saved (from step 170 above) from the Run Name list.
SECTION: iSeq loading and set up
178. Select Start Run to start the pre-run check.
SECTION: iSeq loading and set up
179. Wait about 15 minutes for pre-run checks to complete. The run will start automatically after successful completion. Do not open the door during the pre-run check or during the run because it can cause run failure.
SECTION: iSeq loading and set up
180. If an error occurs during the instrument check, select Retry to redo the check.
SECTION: iSeq loading and set up
181. Unload Consumables.

When sequencing is complete, select Eject Cartridge. The software ejects the used cartridge from the instrument.
SECTION: iSeq loading and set up
182. Remove the cartridge from the tray.
SECTION: iSeq loading and set up
183. Remove the flow cell from the cartridge.
SECTION: iSeq loading and set up
184. Dispose of the flow cell, which has electronic components, in accordance with applicable standards for your institution.
SECTION: iSeq loading and set up
185. Dispose of the cartridge, which contains used reagents, in accordance with applicable standards for your institution.
SECTION: iSeq loading and set up
186. Select Close Door to reload the tray and return to the Home screen. The software automatically reloads the tray and sensors confirm the cartridge removal.
SECTION: iSeq loading and set up
187. Share the Run.

Sign in to BaseSpace on a web browser and check that the run is complete.
SECTION: iSeq loading and set up
188. Click on the run name. Select “Share”, enter the email of the person you would like to share with, and make sure that the check box for sharing the project remains clicked.
SECTION: Analysis
189. Assessment of sequence quality can be performed using the following protocol:
dx.doi.org/10.17504/protocols.io.bdvfi63n
SECTION: References
190. Qiagen DNeasy Blood & Tissue Handbook. HB-2061-003 07/2020
https://www.qiagen.com/us/resources/resourcedetail?id=68f29296-5a9f-40fa-8b3d-1c148d0b3030&lang=en
SECTION: References
191. Illumina DNA Prep documentation
https://support.illumina.com/sequencing/sequencing_kits/illumina-dna-prep/documentation.html
SECTION: References
192. Illumina iSeq 100 Sequencing System Guide
https://support.illumina.com/downloads/iseq-100-system-guide-1000000036024.html
SECTION: References
193. Illumina Index Pooling Guide
https://support-docs.illumina.com/SHARE/IndexAdapterPooling/Content/SHARE/IndexAdapterPooling/Nextera/Pooling_fNXT.htm
SECTION: Spotlight video
194.