TITLE: iSeq Bacterial WGS Protocol
AUTHORS: Sarah Nemser, Laura Goodman, Renee Anerson, Melanie Prarat, Katherine Shiplett

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Whole genome sequencing procedure using Qiagen spin columns for DNA extraction, Illumina DNA Prep (previously called Nextera Flex) for library preparation, and iSeq 100 chemistry for sequencing. This protocol covers from bacterial propagation through loading and running the iSeq machine.</div><br/><div class = "text-block"><span>This protocol is written for running 4 practice </span><span style = "font-style:italic;">Salmonella</span><span> isolates but can be easily adjusted for different numbers of isolates and species. It is recommended to run no more than 4 isolates for an initial practice or training run. Up to 6 isolates may be included if smaller genomes are pooled.</span></div></div>

[STEPS]
?. [Bacterial propagation and DNA extraction]
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.
?. [Bacterial propagation and DNA extraction]
See "guidelines and warnings" for general precautions. Assemble the following materials and reagents:  ABC1Kit and ReagentComponents and notesVolume for 1 Sample 2  Qiagen DNeasy Blood &
  Tissue Kit     Buffer ATL    180 μL  3Proteinase K (Qiagen 19131 or 19133)20 µL – Gram negative bacteria
25 µL – Gram positive bacteria4Buffer AL  200 μL5Buffer ATLfor Gram negative bacteria only180 μL6Buffer AW198mL concentrate+130mL 200 proof ethanol500 μL7Buffer AW266mL concentrate+160mL 200 proof ethanol500 μL8DNeasy additional collection tubes  (Qiagen #19201)Additional tubes recommended to reduce cross-contamination risk from re-using tubes during wash steps3 tubes9  Qiagen #19086    Buffer EB (for EDTA-free elution of samples)    100 μL  10  Enzymatic
  lysis (ELB)-lysozyme mixture for Gram positive isolates only  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; add 3.6 mg lysozyme right before use  180 μL  11  Lysozyme-
  for Gram positive isolates only    (for making ELB)    NA  12  Triton
  X-100- for Gram positive isolates only    (for making ELB)    NA  13  1 M
  Tris-HCl, pH 8.0- for Gram positive isolates only    (for making ELB)    NA  140.5 M EDTA(for making ELB)NA15RNAse A, Qiagen #19101(for optional RNAse treatment of DNA)4 μL16  200 proof or 96-100%
  ethanol, Molecular biology grade    NA    200 μL  17  Water, Molecular biology
  Grade    For resuspending colonies,
  preparing 80% EtOH, and dilution of DNA    200 μL  18  Inoculating
  loop (1 μL) (BD, #220215)    NA
ABC1Kit and ReagentComponents and notesVolume for 1 Sample 2  Qiagen DNeasy Blood &
  Tissue Kit     Buffer ATL    180 μL  3Proteinase K (Qiagen 19131 or 19133)20 µL – Gram negative bacteria
25 µL – Gram positive bacteria4Buffer AL  200 μL5Buffer ATLfor Gram negative bacteria only180 μL6Buffer AW198mL concentrate+130mL 200 proof ethanol500 μL7Buffer AW266mL concentrate+160mL 200 proof ethanol500 μL8DNeasy additional collection tubes  (Qiagen #19201)Additional tubes recommended to reduce cross-contamination risk from re-using tubes during wash steps3 tubes9  Qiagen #19086    Buffer EB (for EDTA-free elution of samples)    100 μL  10  Enzymatic
  lysis (ELB)-lysozyme mixture for Gram positive isolates only  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; add 3.6 mg lysozyme right before use  180 μL  11  Lysozyme-
  for Gram positive isolates only    (for making ELB)    NA  12  Triton
  X-100- for Gram positive isolates only    (for making ELB)    NA  13  1 M
  Tris-HCl, pH 8.0- for Gram positive isolates only    (for making ELB)    NA  140.5 M EDTA(for making ELB)NA15RNAse A, Qiagen #19101(for optional RNAse treatment of DNA)4 μL16  200 proof or 96-100%
  ethanol, Molecular biology grade    NA    200 μL  17  Water, Molecular biology
  Grade    For resuspending colonies,
  preparing 80% EtOH, and dilution of DNA    200 μL  18  Inoculating
  loop (1 μL) (BD, #220215)    NA
?. [Bacterial propagation and DNA extraction]
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.
?. [Bacterial propagation and DNA extraction]
Add 200 μL ethanol (200 proof) to each tube and mix thoroughly by vortexing followed by a brief pulse spin.
?. [Bacterial propagation and DNA extraction]
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.
?. [Bacterial propagation and DNA extraction]
Add 500 μL Buffer AW1, and centrifuge for 1 minute at 6,000xg (8,000rpm). Discard the flow-through and the collection tube.
?. [Bacterial propagation and DNA extraction]
Add 500 μL Buffer AW2, and centrifuge for 1 minute at 6,000xg (8,000rpm). Discard the flow-through and the collection tube.
?. [Bacterial propagation and DNA extraction]
Transfer the spin column to a new collection tube.
?. [Bacterial propagation and DNA extraction]
Centrifuge for 1 minute at 18,000-20,000xg (14,000rpm) to dry.
?. [Bacterial propagation and DNA extraction]
Place the spin column in a clean, labelled 1.5 mL microcentrifuge tube, and pipet 100 μL Buffer EB directly onto the DNeasy membrane.
?. [Bacterial propagation and DNA extraction]
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.
?. [Bacterial propagation and DNA extraction]
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.
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
Assemble the following materials and reagents:  ABCD1Equipment, Kit, and ReagentComponentsAmount per SampleAmount for 4 Samples + 2 Standards and +1 extra2Qubit 2.0, 3.0, or 4.0 Fluorometer  Invitrogen, #: Q32866    NA    NA    NA  3Qubit dsDNA Broad Range (BR) Assay Kit (2-1000ng), #: Q32850Qubit
  dsDNA BR Buffer (store at room temperature)199μL1393 μL4Qubit dsDNA BR Reagent (200X) (store at room temperature protected from light)1 μL7 μL5Qubit dsDNA BR Stanard #1 (store at 4°C)  10 μL  10 μL6Qubit dsDNA BR Standard #2 (store at 4°C)  10 μL  10 μL7Qubit dsDNA High Sensitivity (HS) Assay Kit
  (0.2-100ng) #: Q32854Qubit
  dsDNA HS Buffer (store at room temperature)199 μL1393 μL8Qubit dsDNA HS Reagent (200X) (store at room temperature protected from light)1 μL7 μL9Qubit dsDNA HS Standard #1 (store at 4°C)  10 μL  10 μL10Qubit dsDNA HS Standard #2 (store at 4°C)  10 μL  10 μL11Qubit assay tubes, #: Q32856  NA    NA    NA  12Water, molecular biology grade  For DNA dilution    NA    NA
ABCD1Equipment, Kit, and ReagentComponentsAmount per SampleAmount for 4 Samples + 2 Standards and +1 extra2Qubit 2.0, 3.0, or 4.0 Fluorometer  Invitrogen, #: Q32866    NA    NA    NA  3Qubit dsDNA Broad Range (BR) Assay Kit (2-1000ng), #: Q32850Qubit
  dsDNA BR Buffer (store at room temperature)199μL1393 μL4Qubit dsDNA BR Reagent (200X) (store at room temperature protected from light)1 μL7 μL5Qubit dsDNA BR Stanard #1 (store at 4°C)  10 μL  10 μL6Qubit dsDNA BR Standard #2 (store at 4°C)  10 μL  10 μL7Qubit dsDNA High Sensitivity (HS) Assay Kit
  (0.2-100ng) #: Q32854Qubit
  dsDNA HS Buffer (store at room temperature)199 μL1393 μL8Qubit dsDNA HS Reagent (200X) (store at room temperature protected from light)1 μL7 μL9Qubit dsDNA HS Standard #1 (store at 4°C)  10 μL  10 μL10Qubit dsDNA HS Standard #2 (store at 4°C)  10 μL  10 μL11Qubit assay tubes, #: Q32856  NA    NA    NA  12Water, molecular biology grade  For DNA dilution    NA    NA
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
Label the tops of Qubit assay tubes to correspond to sample label and BR dsDNA standard label. Do not label the tube sides.
?. [2.	DNA quantification and quality check]
Add 190 μL of Qubit working solution to each of the Qubit assay tubes used for Standard #1 and #2.
?. [2.	DNA quantification and quality check]
Add 196 μL Qubit working solution to individual Qubit assay tubes used for each sample.
?. [2.	DNA quantification and quality check]
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).
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
On the Standards screen, press Yes to read the standards.
?. [2.	DNA quantification and quality check]
Insert the tube containing Standard #1 into the sample chamber, close the lid, then press Read. When the reading is complete, remove Standard #1.
?. [2.	DNA quantification and quality check]
Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read. When the reading is complete, remove Standard #2.
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
Repeat reading until all samples have been read.
?. [2.	DNA quantification and quality check]
Record the Qubit readings in the Initial Dilution tab, Column I, DNA Concentration, of the DNA Prep workbook.
?. [2.	DNA quantification and quality check]
DNA quantification and/or quality check using NanoDrop.
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							  ABCD1Equipment, Kit, and ReagentComponentsAmount per SampleAmount for 4 Samples + 2 Standards and +1 extra2NanoDrop (ThermoFisher)  NA    NA    NA
ABCD1Equipment, Kit, and ReagentComponentsAmount per SampleAmount for 4 Samples + 2 Standards and +1 extra2NanoDrop (ThermoFisher)  NA    NA    NA
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
Record the NanoDrop concentration readings in the Initial Dilution tab, Column H, DNA Concentration, of the DNAFlex workbook.
?. [2.	DNA quantification and quality check]
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.
?. [2.	DNA quantification and quality check]
Clean both upper and lower pedestals of sample loading area with water and laboratory wipes. Close the upper arm.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Assemble the following materials and reagents:  ABC1Kit and ReagentComponentsAmount for each sample2Magnet stand, to handle tubes or plates, depending upon sample throughput  NA    NA  3Illumina DNA Prep Library Kit, Box 1 (4 degrees storage)Sample Purification Beads (SPB)45 μL  + 15 μL4Tagment Stop Buffer (TSB)10 µL5Tagment Wash Buffer (TWB) Pipette slowly to minimize foaming100 µL x 36Illumina DNA Prep Library Prep Kit, Box 2 (-20 degrees storage) Tagmentation Buffer 1 (TB1)  10 μL  7Enhanced PCR Mix (EPM)20 µL8Resuspension Buffer (RSB)32 µL9Illumina DNA Prep Library Prep Kit, Box 3 (4 degrees storage)Bead-Linked Transposomes (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  10Ethanol 200 proof for molecular biology (any supplier)Freshly prepared 80% ethanol    180 μL X 2  11i7 and i5 index  Nextera DNA CD Indexes (24 indexes, Cat. no. 20018707, tube format) OR in plate format Cat. no. 20018708i7 indices: H705, H706, H707, H7105 μL each12i5 indices: H503, H505, H506, H5175 μL each13PhiX Control v3, 10nM (Illumina, FC-110-3001)  NA    NA  14  Ice    NA    NA  15  DNA    NA    NA
ABC1Kit and ReagentComponentsAmount for each sample2Magnet stand, to handle tubes or plates, depending upon sample throughput  NA    NA  3Illumina DNA Prep Library Kit, Box 1 (4 degrees storage)Sample Purification Beads (SPB)45 μL  + 15 μL4Tagment Stop Buffer (TSB)10 µL5Tagment Wash Buffer (TWB) Pipette slowly to minimize foaming100 µL x 36Illumina DNA Prep Library Prep Kit, Box 2 (-20 degrees storage) Tagmentation Buffer 1 (TB1)  10 μL  7Enhanced PCR Mix (EPM)20 µL8Resuspension Buffer (RSB)32 µL9Illumina DNA Prep Library Prep Kit, Box 3 (4 degrees storage)Bead-Linked Transposomes (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  10Ethanol 200 proof for molecular biology (any supplier)Freshly prepared 80% ethanol    180 μL X 2  11i7 and i5 index  Nextera DNA CD Indexes (24 indexes, Cat. no. 20018707, tube format) OR in plate format Cat. no. 20018708i7 indices: H705, H706, H707, H7105 μL each12i5 indices: H503, H505, H506, H5175 μL each13PhiX Control v3, 10nM (Illumina, FC-110-3001)  NA    NA  14  Ice    NA    NA  15  DNA    NA    NA
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Check that the DNA input (ng) is 100-500 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: The minimum volume of input DNA is 2 μL. The maximum input volume is 30 μL of DNA.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Add enough nuclease-free water calculated in Column L of the "DNA Flex Workbook" to each sample in a clean PCR tube.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Add volume of DNA adjusted in Column K of the "DNA Flex Workbook" to the sample tubes now containing nuclease-free water.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Mix by pitetting 5-10 times. Close tubes (or seal plate) and store at 4-8º C overnight, or proceed to step 59.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Vortex BLT vigorously for 10 seconds, visually check the beads for complete resuspension and repeat as necessary. Vortex the TB1.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Prepare the tagmentation master mix according to the following table:  ABC1ReagentVolume per SampleFor 4 Samples2TB110 µL40 µL3BLT10 µL40 µL
ABC1ReagentVolume per SampleFor 4 Samples2TB110 µL40 µL3BLT10 µL40 µL
?. [3.	Nextera DNA Flex/Prep Library preparation]
Vortex the Tagmentation master mix thoroughly to make sure the BLT beads are evenly resuspended in the buffer.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Using fresh tips, transfer 20 µL of Tagmentation master mix to each well containing a sample.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Pipette up and down 10 times mixing the 50 µL reaction to resuspend the beads.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Close the PCR tube (or seal the plate with Microseal 'A' or equivalent if using a 96-well format).
?. [3.	Nextera DNA Flex/Prep Library preparation]
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: (50 µL total reaction)1.   55°C for 15 minutes2.   Hold at 10°CUpon completion of the incubation, remove the PCR tubes from the thermalcycler and proceed to Post Tagmentation Cleanup steps.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Post Tagmentation Cleanup. Check for precipitates in the TSB. If present, heat the buffer at 37°C for 10 minutes and vortex until dissolved.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Open the PCR tubes (or remove the seal from plate).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Add 10 µL of TSB to the tagmentation reaction.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Gently pipette up and down 10 times, mixing the entire volume to resuspend the beads.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Close the PCR tubes (or seal the plate with Microseal 'A' or equivalent).
?. [3.	Nextera DNA Flex/Prep Library preparation]
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 60 µL total reaction)1.   37°C for 15 minutes2.   Hold at 10°C
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the EPM and indices from freezer and allow to come to room temperature while this incubation is running to prepare for later steps.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the PCR tubes from the thermalcycler, quick spin down, then open the tubes (or remove the seal).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Place on the magnet for 3 minutes or until solution is clear.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Use a multichannel pipette, remove the supernatant and discard.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Place the tubes on the magnet for 3 minutes or until solution is clear.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the supernatant and discard.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Repeat steps 77 - 79 one more time for a total of two washes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Amplify Tagemented DNA. Invert the EPM to mix.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Briefly spin the Index tubes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Prepare the PCR master mix according to the following table:  ABC1ReagentVolume per SampleFor 4 Samples2EPM20 µL80 µL3Molecular grade water20 µL80 µL
ABC1ReagentVolume per SampleFor 4 Samples2EPM20 µL80 µL3Molecular grade water20 µL80 µL
?. [3.	Nextera DNA Flex/Prep Library preparation]
Vortex and spin down the PCR master mix.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the third TWB wash from the samples while on the magnet. Remove any excess liquid from the tubes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the tubes from the magnet and immediately proceed to adding the master mix.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Add 40 µL of the PCR master mix to each sample well. Gently pipette to mix to ensure beads are resuspended.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Open the i7 primer tubes with orange caps and discard the caps. Add 5 µL of index primers to the NTA tube. After completion, use orange replacement caps to close the primer tubes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Open the i5 primer tubes with white caps and discard the caps. Add 5 µL of index primers to the NTA tube. After completion, use the white replacement caps to close the primer tubes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Close the tubes (or seal the plate with Microseal 'A' or equivalent).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Place the tubes into the thermalcycler and run the following program (FLEXPCR):PCR Conditions: (50 µL total reaction)1.     68°C for 3 minutes2.     98°C for 3 minutes3.     98°C for 45 seconds4.     62°C for 30 seconds5.     68°C for 2 minutes6.     Repeat steps 3-5 an additional 4 times for a total of 5 cycles7.     68°C for 1 minute8.     Hold at 10°C
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the SPB from the refrigerator to equilibrate to room temperature if proceeding to Clean Up Libraries step.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the tubes from the thermal cycler.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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:  ABC1ReagentVolme per SampleFor 4 Samples2100% Ethanol0.4 ml1.6 ml3Molecular grade water0.1 ml0.4 ml
ABC1ReagentVolme per SampleFor 4 Samples2100% Ethanol0.4 ml1.6 ml3Molecular grade water0.1 ml0.4 ml
?. [3.	Nextera DNA Flex/Prep Library preparation]
Be sure the SPB is at room temperature (at least 30 minutes from refrigerator).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Bring RSB to room temperature (from the freezer) and vortex to mix.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Vortex and invert SPB several times to fully re-suspend the beads.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Prepare SPB master mix:  ABC1ReagentVolume per SampleFor 4 Samples2SPB45 µL180 µL3Molecular grad water40 µL160 µLNOTE: It is recommended to increase the number of samples 3-4 to ensure sufficient volume of master mix.
ABC1ReagentVolume per SampleFor 4 Samples2SPB45 µL180 µL3Molecular grad water40 µL160 µL
?. [3.	Nextera DNA Flex/Prep Library preparation]
If PCR tubes (or plate) were retrieved from cold storage, centrifuge at 280 x g for 1 minute.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the seal.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Place the tubes on the magnet for 3-5 minutes (until supernatant is clear).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Transfer 45 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).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the tubes from magnet and discard.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Vortex and then add 85 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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Pipette to mix a minimum of 10 times until thoroughly mixed (use caution as volume is > 100 µL).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Incubate at room temperature for 5 minutes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Place tubes on the magnet for 5 minutes or until supernatant is clear.
?. [3.	Nextera DNA Flex/Prep Library preparation]
During the incubation, vortex the SPB stock tube thoroughly, and add 15 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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Transfer 125 µL of supernatant  to the wells containing the 15 µLSPB (stock concentration).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Gently pipette to mix 10 times at a volume of ~85 µL, avoiding the introduction of air bubbles.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Incubate at room temperature for 5 minutes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Place the tubes on the magnet for 5 minutes or until clear.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove and discard the supernatant without disrupting the beads.
?. [3.	Nextera DNA Flex/Prep Library preparation]
With the tubes on the magnet, add 180 µL of freshly prepared 80% ethanol and incubate for 30 seconds.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Pipette to remove the ethanol supernatant.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Repeat steps 118 and 119 for a total of two washes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Use a pipette to remove any excess liquid from tubes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Leave beads to air dry on the magnet for 5 minutes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Remove the tubes from the magnet and add 32 µLof RSB to the beads.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Pipette to mix until thoroughly resuspended.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Incubate at room temperature for 2-5 minutes.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Place the tubes onto the magnet for 3 minutes until clear.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Transfer 30 µL of the supernatant into a new tube for each sample (or new well on a 96-well plate for each sample).
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Pool 5 μL per sample into a single 1.5ml tube. Optional: for smaller bacterial genomes, reduce the input volume to 2.5  μL.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Mix by vortexing then spin down.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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”.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Dilute the final library to 4 nM according to the values on the workbook using RSB.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Add 5 µl of the 4 nM pool to 15 µL RSB to a new low bind microtube to make 1 nM pool.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Vortex briefly, and then quick-spin at 280 x g for 3 sec.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.  ABC120˚C to 25˚C water bath6 hours, not exceeding 18 hoursUse 6  L water per cartridge.  Set a  temperature-controlled water bath to 25˚C or mix hot and cold water to  achieve 20˚C to 25˚C. Face the bag label up, submerge the cartridge completely,  and apply ~2 kg weight to prevent floating. Do not stack cartridges in the  water bath unless it is temperature-controlled.22˚C to 8˚C refrigerator36 hours, not exceeding 1 weekPosition the cartridge so that the label faces up and air can circulate on all sides, including the bottom.3Room-temperature air (20˚C to 25˚C)9 hours, not exceeding 18 hoursPosition the cartridge so that the label faces up and air can circulate on all sides, including the bottom.If cartridge is wet from a water bath, dry with paper towels.
ABC120˚C to 25˚C water bath6 hours, not exceeding 18 hoursUse 6  L water per cartridge.  Set a  temperature-controlled water bath to 25˚C or mix hot and cold water to  achieve 20˚C to 25˚C. Face the bag label up, submerge the cartridge completely,  and apply ~2 kg weight to prevent floating. Do not stack cartridges in the  water bath unless it is temperature-controlled.22˚C to 8˚C refrigerator36 hours, not exceeding 1 weekPosition the cartridge so that the label faces up and air can circulate on all sides, including the bottom.3Room-temperature air (20˚C to 25˚C)9 hours, not exceeding 18 hoursPosition the cartridge so that the label faces up and air can circulate on all sides, including the bottom.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Vortex briefly, and then centrifuge at 280 x g for 3 sec.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Vortex briefly, and then centrifuge at 280 x g for 3 sec. Set aside on ice.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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.
?. [3.	Nextera DNA Flex/Prep Library preparation]
Vortex briefly, and then centrifuge at 280 x g for 3 sec.
?. [3.	Nextera DNA Flex/Prep Library preparation]
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).
?. [3.	Nextera DNA Flex/Prep Library preparation]
Set aside the library with PhiX spike-in on ice. This is the final loading product.
?. [iSeq loading and set up]
Load the Flow Cell.Avoiding the access window on top of the cartridge, remove the cartridge from the bag. Discard the bag.
?. [Bacterial propagation and DNA extraction]
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.
?. [Bacterial propagation and DNA extraction]
Incubate at 56°C for 30 minutes, vortexing periodically.
?. [Bacterial propagation and DNA extraction]
Optional: Add 4 μL of RNase A to each tube, vortex and then incubate at RT for 3-5 minutes.
?. [Bacterial propagation and DNA extraction]
Add 25 μL Proteinase K and 200 μL Buffer AL to the tube.
?. [Bacterial propagation and DNA extraction]
Mix thoroughly by vortexing, and incubate at 56°C for 30 minutes. Proceed to step 14.
?. [Bacterial propagation and DNA extraction]
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.
?. [Bacterial propagation and DNA extraction]
Incubate at 56°C for a minimum of 1 hour or up to overnight, vortexing periodically.
?. [Bacterial propagation and DNA extraction]
Optional: Add 4 μL of RNase A to each tube, vortex and then incubate at RT for 3-5 minutes.
?. [Bacterial propagation and DNA extraction]
Add 200 μL Buffer AL to the tube.
?. [Bacterial propagation and DNA extraction]
Mix thoroughly by vortexing, and incubate at 56°C for 10 minutes. Proceed to step 14.
?. [2.	DNA quantification and quality check]
Clean the upper and lower pedestals of the sample loading area with a laboratory wipe.
?. [iSeq loading and set up]
Invert the cartridge five times to mix reagents. Internal components can rattle during inversion, which is normal.
?. [iSeq loading and set up]
Tap the cartridge (label facing up) on the bench or other hard surface five times to ensure reagent aspiration.
?. [iSeq loading and set up]
Using a new pipette tip, pierce the Library reservoir and push the foil to the edges to enlarge the hole. Discard the pipette tip.
?. [iSeq loading and set up]
Add 20 µL diluted library to the bottom of the reservoir. Avoid touching the foil.
?. [iSeq loading and set up]
Open the white foil package containing the flow cell from the notches. Use within 24 hours of opening.
?. [iSeq loading and set up]
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.
?. [iSeq loading and set up]
Hold the flow cell by the grip points with the label facing up.
?. [iSeq loading and set up]
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.
?. [iSeq loading and set up]
Set Up a Sequencing Run.From the control software menu, select Local Run Manager, and then select Open Local Run Manager.
?. [iSeq loading and set up]
Select + Create Run.
?. [iSeq loading and set up]
From the three available options, select Generate FASTQ.
?. [iSeq loading and set up]
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].
?. [iSeq loading and set up]
Enter Run Description (e.g. "Practice Samples").
?. [iSeq loading and set up]
Under Run Settings, select the appropriate Library Prep Kit (Nextera DNA CD Indexes-24 index tube.)
?. [iSeq loading and set up]
Index Reads, select the box next to “2.”
?. [iSeq loading and set up]
Read Type, select Paired End.
?. [iSeq loading and set up]
Read Lengths, be sure that 151 has been entered for Read 1.
?. [iSeq loading and set up]
Read Lengths, be sure that 151 has been entered for Read 2.
?. [iSeq loading and set up]
Under Module-Specific Settings, be sure the Adapter Trimming option “On” is selected.
?. [iSeq loading and set up]
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.
?. [iSeq loading and set up]
Under INDEX plate location using the dropdown.
?. [iSeq loading and set up]
Save the run.
?. [iSeq loading and set up]
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.
?. [iSeq loading and set up]
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.
?. [iSeq loading and set up]
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.
?. [iSeq loading and set up]
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.
?. [iSeq loading and set up]
Under configurations, make sure that “run monitoring and storage” has been checked.
?. [iSeq loading and set up]
Select Run Setup.
?. [iSeq loading and set up]
Select a Run and Review Pre-Run Checks.Select the run you saved (from step 170 above) from the Run Name list.
?. [iSeq loading and set up]
Select Start Run to start the pre-run check.
?. [iSeq loading and set up]
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.
?. [iSeq loading and set up]
If an error occurs during the instrument check, select Retry to redo the check.
?. [iSeq loading and set up]
Unload Consumables.When sequencing is complete, select Eject Cartridge. The software ejects the used cartridge from the instrument.
?. [iSeq loading and set up]
Remove the cartridge from the tray.
?. [iSeq loading and set up]
Remove the flow cell from the cartridge.
?. [iSeq loading and set up]
Dispose of the flow cell, which has electronic components, in accordance with applicable standards for your institution.
?. [iSeq loading and set up]
Dispose of the cartridge, which contains used reagents, in accordance with applicable standards for your institution.
?. [iSeq loading and set up]
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.
?. [iSeq loading and set up]
Share the Run.Sign in to BaseSpace on a web browser and check that the run is complete.
?. [iSeq loading and set up]
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.
?. [References]
Illumina DNA Prep documentationhttps://support.illumina.com/sequencing/sequencing_kits/illumina-dna-prep/documentation.html
?. [References]
Qiagen DNeasy Blood & Tissue Handbook. HB-2061-003 07/2020https://www.qiagen.com/us/resources/resourcedetail?id=68f29296-5a9f-40fa-8b3d-1c148d0b3030&lang=en
?. [2.	DNA quantification and quality check]
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.
?. [References]
Illumina iSeq 100 Sequencing System Guidehttps://support.illumina.com/downloads/iseq-100-system-guide-1000000036024.html
?. [References]
Illumina Index Pooling Guidehttps://support-docs.illumina.com/SHARE/IndexAdapterPooling/Content/SHARE/IndexAdapterPooling/Nextera/Pooling_fNXT.htm
?. [Analysis]
Assessment of sequence quality can be performed using the following protocol:dx.doi.org/10.17504/protocols.io.bdvfi63n