TITLE: 16S Amplicon PCR for the V3-V4 region for the MicroCOPD samples
AUTHORS: Tuyen Hoang, Harald Wiker, Tomas Mikal L. Eagan, Christine Drengenes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the protocol for DNA extraction, PCR, and MiSeq sample preparation for the microbiome samples from sputum, and the bronchoscopies in the Bergen COPD Cohort study, Bergen COPD Exacerbation Study, the Bergen COPD Microbiome Study and the Bergen ILD Microbiome Study. </div><div class = "text-block">Slightly modified Illumina protocol for our low-biomass airways samples in the Bergen COPD & ILD studies. </div><div class = "text-block">The protocol that was the template for the PCR and sample preparation is:</div><div class = "text-block"><span style = "font-weight:bold;">16S Metagenomic Sequencing Library Preparation</span></div><div class = "text-block">Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System</div><div class = "text-block">Part # 15044223 Rev.B. </div></div>

[STEPS]
?. [Amplicon PCR]
Select and thaw DNA samples for PCR.
?. [Amplicon PCR]
Create an overview excel sheet and a plate- and pipetting-layout for the PCR run.
?. [Amplicon PCR]
Spin DNA samples at 13000 x g for 1 minute at room temperature.
?. [Amplicon PCR]
Pipette 5 μl of each DNA sample into a well on a PCR plate, following the plate- and pipetting-layout.
?. [Amplicon PCR]
Prepare a master mix containing the following components, in 2 Eppendorf tubes in a prePCR-hood/room. Vortex briefly to mix, and spin briefly to bring down the content.  ABC1ComponentVol (μl) per tubeTotal vol (μl)216S Amplicon F primer (10uM)27.555316S Amplicon R primer (10uM)27.55542X KAPA HiFi HotStart ReadyMix687.513755PCR grade water357.57156Total11002200
ABC1ComponentVol (μl) per tubeTotal vol (μl)216S Amplicon F primer (10uM)27.555316S Amplicon R primer (10uM)27.55542X KAPA HiFi HotStart ReadyMix687.513755PCR grade water357.57156Total11002200
?. [Amplicon PCR]
Combine the master mix from the two Eppendorf-tubes in a sterile trough. Using a multichannel pipette, transfer 20 μl to each well of the PCR plate. Pipette up and down 10 times to mix.
?. [Amplicon PCR]
Seal the PCR plate with MicroAmp film.
?. [Amplicon PCR]
Centrifuge the PCR plate at 1000 x g for 1 minute at 20 °C.
?. [Amplicon PCR]
Run PCR in a thermal cycler using the following program:  ABC195°C3 min295°C30 sec45 cycles, row 2->4355°C30 sec472°C30 sec572°C5 min64°Cindef
ABC195°C3 min295°C30 sec45 cycles, row 2->4355°C30 sec472°C30 sec572°C5 min64°Cindef
?. [Amplicon PCR]
Continue with Agarose Gel Electrophoresis for checking of 16S PCR amplicons or store PCR plate at -20°C until use.
?. [Agarose electrophoresis]
Thaw and spin down 16S amplicons in Amplicon PCR plate at 1000 x g at 20°C for 1 minute.
?. [Agarose electrophoresis]
Cast two big 1.5% agarose gels containing GelRed (per gel use: 3g agarose + 200 ml 1xTAE + 10 μl GelRed). Use four 15-lane combs per gel.
?. [Agarose electrophoresis]
On a new 96-well plate, add 1 μl 5x DNA loading buffer (use a multichannel pipette and pipette from buffer aliquots in 8 PCR tubes).
?. [Agarose electrophoresis]
Using a multichannel pipette, transfer 5 μl of 16S amplicons from the Amplicon PCR plate to the 96-well plate containing 5x DNA loading buffer. Pipette up and down gently to mix.
?. [Agarose electrophoresis]
Centrifuge plate briefly (10 seconds at 1000 x g) to bring down the content.
?. [Agarose electrophoresis]
Load EZ 100bp ladder (3 µl) on the first lane of each lane-row.
?. [Agarose electrophoresis]
Load samples using a multichannel pipette (remember to note down the sample order in loading).
?. [Agarose electrophoresis]
Run gel in 1xTAE buffer at 80V for 15 minutes.
?. [Agarose electrophoresis]
Increase to 100V and run until the bromophenol blue marker has moved ca. 2/3 of the gel (i.e. 1 hour).
If 1% is used, shorten running time!
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Consumables:  ABC1ItemQuantity (per 96 samples)Storage210 mM Tris pH 8.5 (EB buffer)6 ml-15°C to -25°C3AMPure XP Beads2.5 ml2°C - 8°C4Freshly Prepared 80% Ethanol (EtOH)50 ml596-well 0.2 ml PCR plate2 plates6Microseal 'B' film7Sterile trough810 μl pipette tips with filter3 boxes9200 μl pipette tips with filter7 boxesPreparation: Bring the AMPure XP beads to room temperature.
ABC1ItemQuantity (per 96 samples)Storage210 mM Tris pH 8.5 (EB buffer)6 ml-15°C to -25°C3AMPure XP Beads2.5 ml2°C - 8°C4Freshly Prepared 80% Ethanol (EtOH)50 ml596-well 0.2 ml PCR plate2 plates6Microseal 'B' film7Sterile trough810 μl pipette tips with filter3 boxes9200 μl pipette tips with filter7 boxes
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Centrifuge the Amplicon PCR plate at 1,000 × g at 20°C for 1 minute to collect condensation, carefully remove seal.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Vortex the AMPure XP beads for 30 seconds to make sure that the beads are evenly dispersed. Add an appropriate volume of beads to a trough depending on the number of samples processing (ca 2.5 ml for 96 samples).
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Using a multichannel pipette, add 20 μl of AMPure XP beads to each well of the Amplicon PCR plate, and gently pipette entire volume up and down 10 times. Change tips between columns.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Incubate at room temperature without shaking for 5 minutes.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
With the Amplicon PCR plate on the magnetic stand, use a 100 μl-multichannel pipette to remove first 30 μl and then a 10 μl-multichannel pipette to remove 10 μl and discard the supernatant. Change tips between samples.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
With the Amplicon PCR plate on the magnetic stand, wash the beads with freshly prepared 80% ethanol as follows:a. Using a multichannel pipette, add 200 μl of freshly prepared 80% ethanol to each sample well.b. Incubate the plate on the magnetic stand for 30 seconds.c. Carefully remove and discard 195 μl the supernatant.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
With the Amplicon PCR plate on the magnetic stand, perform a second ethanol wash as follows:a. Using a multichannel pipette, add 200 μl of freshly prepared 80% ethanol to each sample well.b. Incubate the plate on the magnetic stand for 30 seconds.c. Carefully remove and discard 195 μl the supernatant. Use a 10 μl-multichannel pipette to remove the rest of supernatant and excess ethanol.
(Check for excess ethanol in each well by lifting up the plate from magnetic stand and looking from the bottom of each well. Try to remove all supernatant from wells.)
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
With the Amplicon PCR plate still on the magnetic stand, allow the beads to air‐dry for 10 minutes.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Remove the Amplicon PCR plate from the magnetic stand. Using a multichannel pipette, add 52.5 μl of 10 mM Tris pH 8.5 to each well of the Amplicon PCR plate.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Leave stand for 1-2 minutes, and then gently pipette mix up and down 10 times, changing tips after each column. Make sure that beads are fully resuspended.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Incubate at room temperature for 2 minutes.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Place the plate on the magnetic stand for 2 minutes or until the supernatant has cleared.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Using a multichannel pipette, carefully transfer 45 μl of the supernatant from the Amplicon PCR plate to a new 96‐well PCR plate. Change tips between samples to avoid cross‐contamination. Avoid taking AMPure beads along when pipetting the supernatant.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Centrifuge the plate at 1000 x g at 20°C for 1 minute (to bring down the content and remove bubbles).
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Transfer 5 μl of the cleaned-up PCR amplicons to a new 96‐well PCR plate for Index PCR in next steps.
?. [PCR Clean-Up to purify the 16S V3 & V4 amplicon away from free primers and primer dimer species]
Seal the plate with the rest with Microseal “B” adhesive seal and store at -20°C.
Safe stopping point.If you do not immediately proceed to Index PCR, seal the plate with 5 µl amplicons with Microseal "B" adhesive seal and store it at 4°C for short-term (1 day) or at -15°C to -25°C until use (up to a week)
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Consumables:  ABC1ItemQuantity (for 96 samples)Storage22x KAPA HiFi HotStart ReadyMix2 x 1325 µl-15°C to -25°C3Nextera XT Index 1 Primers (N7XX) from the Nextera XT Index kit96 x 5 µl-15°C to -25°C4Nextera XT Index 2 Primers (S5XX) from the Nextera XT Index kit96 x 5 µl-15°C to -25°C5PCR grade water2 x 530 µl-15°C to -25°C6TruSeq Index Plate Fixture (FC-130-1005)17MicroAmp Adhesive Clear Film18Sterile trough3910 μl pipette tips with filter2 boxes +10200 μl pipette tips with filter1 box +
ABC1ItemQuantity (for 96 samples)Storage22x KAPA HiFi HotStart ReadyMix2 x 1325 µl-15°C to -25°C3Nextera XT Index 1 Primers (N7XX) from the Nextera XT Index kit96 x 5 µl-15°C to -25°C4Nextera XT Index 2 Primers (S5XX) from the Nextera XT Index kit96 x 5 µl-15°C to -25°C5PCR grade water2 x 530 µl-15°C to -25°C6TruSeq Index Plate Fixture (FC-130-1005)17MicroAmp Adhesive Clear Film18Sterile trough3910 μl pipette tips with filter2 boxes +10200 μl pipette tips with filter1 box +
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Centrifuge the PCR plate with 5 μl of 16S PCR amplicon after clean-up, at 1000 x g at 20°C for 1 minute.
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Place the plate on a TruSeq Index Plate Fixture (as shown in Fig. 4 below).
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Spin tubes containing Index primers briefly (1 second in a minicentrifuge) to bring down the content (use 2 ml-tubes without caps as “holding adapters”).
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Arrange the Index 2 primer tubes (white caps, clear solution) vertically, aligned with rows A through H in the TruSeq Index Plate Fixture (Fig. 4). Using a multichannel pipette, pipette 5 µl of Index 2 primers to the wells on the PCR plate.
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Arrange the Index 1 primer tubes (orange caps, yellow solution) horizontally, aligned with columns 1 through 12. Using a multichannel pipette, pipette 5 µl of Index 1 primers to the wells on the PCR plate.
For pipetting convenience, begin first with the first 6 primer tubes aligned with columns 1-6 only. Then remove these, and continue with the next 6 primers tubes aligned with columns 7-12
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Prepare a master mix in two 2 ml-tubes, containing:  AB1ComponentVolume per tube (µl)22 x KAPA HiFi HotStart Ready Mix1325 µl3PCR Grade water530 µl4Total1855 µlVortex to mix and spin down.
AB1ComponentVolume per tube (µl)22 x KAPA HiFi HotStart Ready Mix1325 µl3PCR Grade water530 µl4Total1855 µl
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Transfer the master mix to a sterile trough, and pipette 35 µl to each well on the PCR plate using a multichannel pipette. Gently pipette up and down 10 times to mix.
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Cover the plate with MicroAmp Adhesive Clear film.
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Centrifuge the plate at 1,000 × g at 20°C for 1 minute.
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Perform PCR on a thermal cycler using the following program:  ABC195°C3 min295°C30 sec8 cycles, row 2 -> 4355°C30 sec472°C30 sec572°C5 min64°Cindef
ABC195°C3 min295°C30 sec8 cycles, row 2 -> 4355°C30 sec472°C30 sec572°C5 min64°Cindef
?. [Index PCR - attaches dual indices and Illumina sequencing adapters using the Nextera XT Index Kit.]
Store Index PCR plate at -20°C until the next steps.
?. [PCR Clean-Up for Index PCR Samples]
Consumables:  ABC1ItemQuantity (per 96 samples)Storage210 mM Tris pH 8.5 (EB buffer)3 ml-15°C to -25°C3AMPure XP beads6 ml2°C to 8°C4Freshly Prepared 80% Ethanol (EtOH)50 ml596-well 0.2 ml PCR plate3 plates6Microseal 'B' film7Sterile trough4810 µl pipette tips with filter3 boxes9200 µl pipette tips with filter7 boxesPreparation: Bring the AMPure XP beads to room temperature.
ABC1ItemQuantity (per 96 samples)Storage210 mM Tris pH 8.5 (EB buffer)3 ml-15°C to -25°C3AMPure XP beads6 ml2°C to 8°C4Freshly Prepared 80% Ethanol (EtOH)50 ml596-well 0.2 ml PCR plate3 plates6Microseal 'B' film7Sterile trough4810 µl pipette tips with filter3 boxes9200 µl pipette tips with filter7 boxes
?. [PCR Clean-Up for Index PCR Samples]
Centrifuge the Index PCR plate at 280 × g at 20°C for 1 minute to collect condensation.
?. [PCR Clean-Up for Index PCR Samples]
Vortex the AMPure XP beads for 30 seconds to make sure that the beads are evenly dispersed. Add an appropriate volume of beads to a trough (ca 6 ml for 96 samples).
?. [PCR Clean-Up for Index PCR Samples]
Using a multichannel pipette, add 56 μl of AMPure XP beads to each well of the Index PCR plate and gently pipette entire volume up and down 10 times. Change tips between columns.
?. [PCR Clean-Up for Index PCR Samples]
Incubate at room temperature without shaking for 5 minutes.
?. [PCR Clean-Up for Index PCR Samples]
Place the plate on a magnetic stand for 2 minutes or until the supernatant has cleared.
?. [PCR Clean-Up for Index PCR Samples]
With the Amplicon PCR plate on the magnetic stand, use a 100 µl-multichannel pipette to remove first 90 µl and then a 10 µl-multichannel pipette to remove 10 µl and discard the supernatant. Change tips between samples.
?. [PCR Clean-Up for Index PCR Samples]
With the Amplicon PCR plate on the magnetic stand, wash the beads with freshly prepared 80% ethanol as follows:a. Using a multichannel pipette, add 200 μl of freshly prepared 80% ethanol to each sample well.b. Incubate the plate on the magnetic stand for 30 seconds.c. Carefully remove and discard 195 µl the supernatant.
?. [PCR Clean-Up for Index PCR Samples]
With the Amplicon PCR plate on the magnetic stand, perform a second ethanol wash as follows:a. Using a multichannel pipette, add 200 μl of freshly prepared 80% ethanol to each sample well.b. Incubate the plate on the magnetic stand for 30 seconds.c. Carefully remove and discard 195 µl the supernatant.d. Use a 10 µl-multichannel pipette to remove the rest of supernatant and excess ethanol. (Check for excess ethanol in each well by lifting up the plate from magnetic stand and looking from the bottom of each well. Try to remove all supernatant from wells.)
?. [PCR Clean-Up for Index PCR Samples]
With the Index PCR plate still on the magnetic stand, allow the beads to air‐dry for 10 minutes.
?. [PCR Clean-Up for Index PCR Samples]
Remove the Index PCR plate from the magnetic stand. Using a multichannel pipette, add 27.5 μl of 10 mM Tris pH 8.5 to each well of the Index PCR plate.
?. [PCR Clean-Up for Index PCR Samples]
Leave stand for 1-2 min, and then gently pipette mix up and down 10 times, changing tips after each column. Make sure that beads are fully resuspended.
?. [PCR Clean-Up for Index PCR Samples]
Incubate at room temperature for 2 minutes.
?. [PCR Clean-Up for Index PCR Samples]
Place the plate on the magnetic stand for 2 minutes or until the supernatant has cleared.
?. [PCR Clean-Up for Index PCR Samples]
Using a multichannel pipette, carefully transfer 10 μl of the supernatant from the Index PCR plate to a new 96‐well PCR plate. Label this plate with “first plate”. Change tips between samples to avoid cross-contamination.
?. [PCR Clean-Up for Index PCR Samples]
Repeat step 109 (to obtain 2 PCR plates with 10 μl each Index PCR per plate). Label the plate with “second plate”. Change tips between samples to avoid cross-contamination.
Avoid taking XP beads along when transferring the samples
?. [PCR Clean-Up for Index PCR Samples]
Seal (with Microseal B film) and store the plate with minimal XP beads (usually “first plate”) at -20°C for library normalization (dilution) and pooling for use in sequencing with MiSeq.
?. [PCR Clean-Up for Index PCR Samples]
Use the second plate for assessment of DNA concentration and analysis with BioAnalyzer (if not proceeding to these steps right after, seal and store the plate at -20°C until use).
?. [Library Quantification, Normalization & Pooling]
Calculate DNA concentration (C) in each Index PCR sample using the corresponding average concentration value (PicoGreenAve) ng/µl obtained from PicoGreen.C (ng/µl) = PicoGreen Ave x 100
We use Qubit!
?. [Library Quantification, Normalization & Pooling]
Convert concentrations in ng/µl to nM, using the following formula:[(concentration in ng/μl)/(660 mol × 630)]   × 106=  concentration in nM
?. [Library Quantification, Normalization & Pooling]
For normalization, dilute concentrated final library (i.e. index PCR) using 10 mM Tris pH 8.5 (EB buffer) to 4nM.i.	Transfer a desired volume (e.g. 5 µl) of cleaned index PCR samples from the Index PCR plate with minimal contaminated XP beads after clean-up to a new PCR plate.ii.	Add an appropriate amount of EB buffer to each well to make a dilution to 4nM. Pipette up and down gently 5 times to mix.iii.	Label the plate with diluted index PCR samples with “IndexPCR” PLATE #, and “4nM”.
?. [Library Quantification, Normalization & Pooling]
Aliquot 5 µl of each diluted index PCR sample and mix aliquots in an Eppendorf tube for pooling libraries.
?. [Library Quantification, Normalization & Pooling]
Make 5 5µl-aliquots of the library pool, and store at -20°C until use in sample preparation for MiSeq sequencing.
?. [Sample Preparation for MiSeq Sequencing - preparation]
Create a txt/excel file containing the following info separated by tab/in column:  ABCDEFGHIJ1Sample_IDSample_NameSample_PlateSample_WellI7_Index_IDindexI5_Index_IDindex2Sample_ProjectDescription* Tip: use a previous sample sheet as a template and copy over info for the following fields:  ABCD1I7_Index_IDindexI5_Index_IDindex2(index order is meant to be the same for all runs)* If there is any notice about samples (DNA extraction, PCR product, etc), use the Description field* Leave empty fields: Sample_Plate, Sample_Well, and Sample_Project
ABCDEFGHIJ1Sample_IDSample_NameSample_PlateSample_WellI7_Index_IDindexI5_Index_IDindex2Sample_ProjectDescription
ABCD1I7_Index_IDindexI5_Index_IDindex2
?. [Sample Preparation for MiSeq Sequencing - preparation]
Thawing the MiSeq Reagent Cartridge
An alternative to the thawing steps is to thaw reagents overnight in 2°C to 8°C storage. Reagents are stable up to one week when stored at this temperature.
?. [Sample Preparation for MiSeq Sequencing - preparation]
Remove the reagent cartridge from -15°C to -25°C storage.
?. [Sample Preparation for MiSeq Sequencing - preparation]
Place the reagent cartridge in a water bath containing enough room temperature deionized water to submerge the base of the reagent cartridge. Do not allow the water to exceed the maximum water line printed on the reagent cartridge.
?. [Sample Preparation for MiSeq Sequencing - preparation]
Allow the reagent cartridge to thaw in the room temperature water bath for approx 60-90 minutes or until it is thawed completely.
?. [Sample Preparation for MiSeq Sequencing - preparation]
Remove the cartridge from the water bath and gently tap it on the bench to dislodge water from the base of the cartridge. Dry the base of the cartridge.
?. [Sample Preparation for MiSeq Sequencing - preparation]
Invert the reagent cartridge ten times to mix the thawed reagents, and then visually inspect that all positions are thawed.
?. [Sample Preparation for MiSeq Sequencing - preparation]
Visually inspect the reagents in positions 1,2, and 4 to make sure that they are fully mixed and free of precipitates.
?. [Sample Preparation for MiSeq Sequencing - preparation]
Gently tap the cartridge on the bench to reduce air bubbles in the reagents.
The MiSeq sipper tubes go to the bottom of each reservoir to aspirate the reagents, so it is important that the reservoirs are free of air bubbles.
?. [Sample Preparation for MiSeq Sequencing - preparation]
Place the reagent cartridge on ice or set aside at 2°C to 8°C (up to 6 hours) until ready to set up the run. For best results, proceed directly to loading the sample and setting up the run.
?. [Library Denaturing and MiSeq Sample Loading]
Consumables  ABC1ItemQuantityStorage210 mM Tris pH 8.5 (EB buffer, Qiagen)6 µl-15°C to -25°C3HT1 (Hybridization buffer)1540 µl-15°C to -25°C (attached to the MiSeq reagent cartridge)40.2 N NaOH (less than a week old)10 µl-15°C to -25°C5PhiX Control Kit v3 (FC-110-3001)4 µl-15°C to -25°C6MiSeq reagent cartridge1 cartridge-15°C to -25°C71.7 ml microcentrifuge tubes (screw cap recommended)3 tubes82.5 L ice bucket9
ABC1ItemQuantityStorage210 mM Tris pH 8.5 (EB buffer, Qiagen)6 µl-15°C to -25°C3HT1 (Hybridization buffer)1540 µl-15°C to -25°C (attached to the MiSeq reagent cartridge)40.2 N NaOH (less than a week old)10 µl-15°C to -25°C5PhiX Control Kit v3 (FC-110-3001)4 µl-15°C to -25°C6MiSeq reagent cartridge1 cartridge-15°C to -25°C71.7 ml microcentrifuge tubes (screw cap recommended)3 tubes82.5 L ice bucket9
?. [Library Denaturing and MiSeq Sample Loading]
Set a heat block suitable for 1.7 ml microcentrifuge tubes to 96°C.
?. [Library Denaturing and MiSeq Sample Loading]
Prepare 0.2 N NaOH by diluting 2 µl 10M NaOH in 98 µl PCR-grade H2O (total volume = 100 µl).
?. [Library Denaturing and MiSeq Sample Loading]
In an ice bucket, prepare an ice‐water bath by combining 3 parts ice and 1 part water.
?. [Library Denaturing and MiSeq Sample Loading]
Thaw a tube with 5 µl pooled library on ice.
?. [Library Denaturing and MiSeq Sample Loading]
Thaw HT1 (Hybridization Buffer) at room temperature. Keep HT1 on ice (to chill) after thawing.
?. [Library Denaturing and MiSeq Sample Loading]
Combine the following volumes of pooled final DNA library and freshly diluted 0.2 N NaOH in a microcentrifuge tube: • 4 nM pooled library (5 μl) • 0.2 N NaOH (5 μl)
?. [Library Denaturing and MiSeq Sample Loading]
Set aside the remaining dilution of 0.2 N NaOH to prepare a PhiX control within the next 12 hours.
?. [Library Denaturing and MiSeq Sample Loading]
Vortex briefly to mix the sample solution, and then centrifuge the sample solution at 280 × g at 20°C for 1 minute.
?. [Library Denaturing and MiSeq Sample Loading]
Incubate for 5 minutes at room temperature to denature the DNA into single strands.
?. [Library Denaturing and MiSeq Sample Loading]
Add the following volume of pre‐chilled HT1 to the tube containing denatured DNA:• Denatured DNA (10 μl)• Pre‐chilled HT1 (990 μl) Adding the HT1 results in a 20 pM denatured library in 1 mM NaOH.
?. [Library Denaturing and MiSeq Sample Loading]
Place the denatured DNA on ice until you are ready to proceed to final dilution.
?. [Library Denaturing and MiSeq Sample Loading]
Dilute the denatured DNA to 10 pM by combining:• 20 pM denatured library (300 μl)• Pre‐chilled HT1 (300 µl)
?. [Library Denaturing and MiSeq Sample Loading]
Invert several times to mix and then pulse centrifuge the DNA solution.
?. [Library Denaturing and MiSeq Sample Loading]
Place the denatured and diluted DNA on ice.
?. [Library Denaturing and MiSeq Sample Loading]
Combine the following volumes to dilute the PhiX library to 4 nM:• 10 nM PhiX library (2 μl)• 10 mM Tris pH 8.5 (3 μl)
?. [Library Denaturing and MiSeq Sample Loading]
Combine the following volumes of 4 nM PhiX and 0.2 N NaOH in a microcentrifuge tube:• 4 nM PhiX library (5 μl)• 0.2 N NaOH (5 μl)
?. [Library Denaturing and MiSeq Sample Loading]
Vortex briefly to mix the 2 nM PhiX library solution.
?. [Library Denaturing and MiSeq Sample Loading]
Incubate for 5 minutes at room temperature to denature the PhiX library into single strands.
?. [Library Denaturing and MiSeq Sample Loading]
Add the following volumes of pre‐chilled HT1 to the tube containing denatured PhiX library to result in a 20 pM PhiX library:• Denatured PhiX library (10 μl)• Pre‐chilled HT1 (990 μl)
?. [Library Denaturing and MiSeq Sample Loading]
Dilute the denatured 20 pM PhiX library to 10 pM as follows:• 20 pM denatured PhiX library (300 µl)• Pre‐chilled HT1 (300 μl)
?. [Library Denaturing and MiSeq Sample Loading]
Invert several times to mix and then pulse centrifuge the DNA solution.
?. [Library Denaturing and MiSeq Sample Loading]
Place the denatured and diluted PhiX on ice.
?. [Library Denaturing and MiSeq Sample Loading]
Combine the following volumes of denatured PhiX control library and the denatured amplicon library in a microcentrifuge tube:• Denatured and diluted PhiX control (90 μl) – i.e 15% of a total vol of 600µl• Denatured and diluted amplicon library (510 μl)
15% PhiX control spike‐in is used
?. [Library Denaturing and MiSeq Sample Loading]
Set the combined sample library and PhiX control aside on ice until you are ready to heat denature the mixture immediately before loading it onto the MiSeq v3 reagent cartridge.
?. [Library Denaturing and MiSeq Sample Loading]
Using a heat block, incubate the combined library and PhiX control tube at 96°C for 2 minutes.
Perform the heat denaturation step immediately before loading the library into the MiSeq reagent cartridge to ensure efficient template loading on the MiSeq flow cell
?. [Library Denaturing and MiSeq Sample Loading]
After the incubation, invert the tube 1–2 times to mix and immediately place in the icewater bath.
?. [Library Denaturing and MiSeq Sample Loading]
Keep the tube in the ice‐water bath for 5 minutes.
?. [DNA extraction]
Reagents:  ABC1ReagentSupplierCatalog Number2Sputasol stockOxoidSR02333Molecular grade H2OMerckH2OMB10014Lysozyme (10 mg/mL)Sigma-AldrichL3790 - 10 x 1 mL5Mutanolysin (25,000 U/mL)Sigma-AldrichM9901-50KU6Lysostaphin (4000 U/mL)Sigma-AldrichL4402-5MG7FastDNA SPIN kitMP Biomedicals6540-600
ABC1ReagentSupplierCatalog Number2Sputasol stockOxoidSR02333Molecular grade H2OMerckH2OMB10014Lysozyme (10 mg/mL)Sigma-AldrichL3790 - 10 x 1 mL5Mutanolysin (25,000 U/mL)Sigma-AldrichM9901-50KU6Lysostaphin (4000 U/mL)Sigma-AldrichL4402-5MG7FastDNA SPIN kitMP Biomedicals6540-600
?. [DNA extraction]
Take the samples out from the -80 °C freezer and leave to thaw at room temperature.   ABC1SampleAbbreviationVolume for extraction (µl)2Negative control PBSCTRL450 µl3Oral washOW1800 µl4Protected bronchoalveolar lavagePBAL (PBAL1 & PBAL2)1800 µl5Protected sterile brushPSB (rPSB & lPSB)450 µl6Small volume lavageSVL1800 µl
ABC1SampleAbbreviationVolume for extraction (µl)2Negative control PBSCTRL450 µl3Oral washOW1800 µl4Protected bronchoalveolar lavagePBAL (PBAL1 & PBAL2)1800 µl5Protected sterile brushPSB (rPSB & lPSB)450 µl6Small volume lavageSVL1800 µl
?. [DNA extraction]
Vortex the samples for 10 seconds and spin down briefly.
?. [DNA extraction]
Transfer 450 µL of the CTRL sample to a sterile 1.5 mL eppendorf tube.
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Split the 1800 µL OW sample into three aliquots of 600 µL each and transfer to sterile 1.5 mL eppendorf tubes.
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Split the 1800 µL PBAL sample into three aliquots of 600 µL each and transfer to sterile 1.5 mL eppendorf tubes.
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Prepare a working solution of Sputasol according to the number of samples being processed:  ABCDEF1# of samples:7-89-1213-1617-2021-242Sputasol stock (ml)0.7501.1251.5001.8752.2503Molecular grade H2O (ml)9.25013.87518.50023.12527.7504Total (ml)1015202530
ABCDEF1# of samples:7-89-1213-1617-2021-242Sputasol stock (ml)0.7501.1251.5001.8752.2503Molecular grade H2O (ml)9.25013.87518.50023.12527.7504Total (ml)1015202530
?. [DNA extraction]
Add 450 µL of the Sputasol working solution to the CTRL sample prepared in step 4. Mix by vortexing for 5 seconds.
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Add 600 µL of the Sputasol working solution to each of the three aliquots of OW sample prepared in step 5. Mix by vortexing for 5 seconds.
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Add 600 µL of the Sputasol working solution to each of the three aliquots of PBAL sample prepared in step 6. Mix by vortexing for 5 seconds.
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Incubate the samples on a thermoshaker at 37°C with shaking at 1000 rpm for 15 minutes.
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To pellet the bacterial cells, centrifuge the samples at 15700 x g at room temperature for 8 minutes.
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Discard the supernatant from each sample, taking care not to disturb the pellet.
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Resuspend the pellet in the CTRL sample with 250 µL PBS.
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Carefully pipette the entire volume up and down 10 times to mix.
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Resuspend and combine the three pellets from the OW sample into one eppendorf tube in a total of 250 µL PBS.
?. [DNA extraction]
Carefully pipette the entire volume up and down 10 times to mix.
?. [DNA extraction]
Resuspend and combine the three pellets from the PBAL sample into one eppendorf tube in a total of 250 µL PBS.
?. [DNA extraction]
Carefully pipette the entire volume up and down 10 times to mix.
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To ensure proper mixing, vortex each sample for 15 seconds and spin down briefly.
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Prepare an enzyme cocktail solution according to the number of samples being processed:  AB1ComponentsVolume/Sample (µl)2Lysozyme (10 mg/ml)253Mutanolysin (25 000 U/ml)34Lysostaphin ( 4 000 U/ml in NaAC)1.55TE5 buffer (10 mM Tris HCl, 5mM EDTA, pH 8.0)20.56Total50
AB1ComponentsVolume/Sample (µl)2Lysozyme (10 mg/ml)253Mutanolysin (25 000 U/ml)34Lysostaphin ( 4 000 U/ml in NaAC)1.55TE5 buffer (10 mM Tris HCl, 5mM EDTA, pH 8.0)20.56Total50
?. [DNA extraction]
Add 50 µl of the enzyme-cocktail solution to each sample. Mix by pipetting the entire volume up and down 10 times. Vortex samples for 1 second.
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Incubate the samples on a thermomixer at 37°C with shaking at 350 rpm for 1 hour.
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Centrifuge the samples at 15700 x g at room temperature for 15 minutes to pellet any bacterial cells not lysed.
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For each sample, transfer the supernatant containing bacterial DNA (about 300 µL) to a separate 2 mL eppendorf tube and store at 4°C. Proceed with pellet.
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Resuspend the pellet in 800 µL CLS-TC buffer (FastDNA SPIN kit) by pipetting up and down 10 times.
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For each sample, transfer the suspension from step 26 to a Lysing Matrix A tube (FastDNA SPIN kit).
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Homogenize the samples in the FastPrep-24 instrument at a speed setting of 6.0 m/s for 40 seconds.
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Centrifuge the samples at 14000 x g at room temperature for 10 minutes.
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Carefully pipette out 650 µL of the supernatant and transfer to the corresponding 2 mL eppendorf tube set aside in step 25. The total volume is now about 950 µL per sample (650 µL + 300 µL).
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Add 950 µL Binding Matrix (FastDNA SPIN kit) to each sample and invert to mix.
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Place the samples on a rotator and incubate for 5 minutes at room temperature at low speed.
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Transfer 700 µl of each suspension to a SPIN filter (FastDNA SPIN kit).
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Centrifuge the SPIN filter at 14000 x g for 1 minute.
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Discard flow-through.
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Repeat steps 33-35 until the whole sample has been spun through the SPIN filter.
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Add 500 µl prepared SEWS-M (FastDNA SPIN kit) and resuspend each pellet by pipetting up and down several times.
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Centrifuge at 14000 x g for 1 minute. Discard flow-through.
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Without adding any liquid, centrifuge the SPIN filters a second time at 14000 x g for 2 minutes.
?. [DNA extraction]
Replace the catch tube with a new catch tube.
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Add 100 μL of DES (FastDNA SPIN kit) and gently resuspend the Binding Matrix by pipetting up and down several times.
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Incubate at 55°C for 5 minutes on a heating block.
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Centrifuge at 14000 x g for 1 minute to elute the DNA.
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Check the purity of the DNA sample using Nanodrop (260/280).
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Measure the DNA concentration using the Qubit fluorometer.