SOP

I. Collection of Clinical Specimens and Processing

1. Stool

1.1 Raise the toilet seat. Place the stool collection frame on the back of the toilet bowl. Place collection bowl in frame. Place toilet seat down. Do not urinate into the collection container. Deposit your stool directly into the collection container.
1.2 Wash your hands and wear gloves. Collect the sample with a spoon (10 g can be dispensed per tube), tighten the lid after sampling. Place the closed container into the Ziploc bag and seal the bag. Write the date and time of collection on the outer bag label.
1.3 Place the sealed Ziploc bag containing your stool specimen on top of the frozen gel packs in the Styrofoam container.
1.4 The container was brought back to the laboratory within 24 hours of collection. If you do not operate immediately, placed at -80 ℃ preservation, to avoid repeated freezing and thawing.

2. Sputum

2.1 Can keep their own sputum: warm water mouthwash, observe whether the food residue, beat back or stimulate the cough, deep breath several times, then cough out of the sputum in the trachea deep to the collector, close the cap.
2.2 Artificial auxiliary breathing: wearing sterile gloves, the sputum collector connected to the negative pressure suction device, open the suction switch, place the catheter inserted into the throat deep, leaving the sputum specimens 5-10ml , close the cap.
2.3 The container was brought back to the laboratory within 2 hours of collection. If you do not operate immediately, placed at -80 °C preservation.

3. Blood

20 ml (2 x 10 ml) will be collected in the Coriell-provided yellow-top collection tubes (vacutainer with ACD Solution A), 2 hours within the inspection.

II. Extraction of Bacterial Genomic DNA

1. Stool

  •  Notes before starting
    a. Prepare a water bath at 70°C for use in steps 3 and 8.
    b. Perform all centrifugation steps at room temperature (15–25°C) at 20,000 x g (~14,000 rpm).
    c. Redissolve any precipitates in Buffer AL and InhibitEX® Buffer by heating and mixing.
    d. Add ethanol to Buffer AW1 and Buffer AW2 concentrates.
  •  Experimental steps
    Materials:QIAamp® Fast DNA Stool Mini Kit
    Procedures:
    a. Weigh 180–220 mg stool in a 2 ml microcentrifuge tube (not provided) and place tube on ice.
    b. Add 1 ml InhibitEX Buffer to each stool sample. Vortex continuously for 1 min or until the stool sample is thoroughly homogenized.
    c. Heat the suspension for 5 min at 70°C. (The lysis temperature can be increased to 95°C for cells that are difficult to lyse.) Vortex for 15 s.
    d. Centrifuge sample for 1 min to pellet stool particles.
    e. Pipet 15 μl Proteinase K into a new 1.5 ml microcentrifuge tube (not provided).
    f. Pipet 200 μl supernatant from step 4 into the 1.5 ml microcentrifuge tube containing Proteinase K.
    g. Add 200 μl Buffer AL and vortex for 15 s. Note: Do not add Proteinase K directly to Buffer AL. It is essential that the sample and Buffer AL are thoroughly mixed to form a homogeneous solution.
    h. Incubate at 70°C for 10 min.
    i. Add 200 μl of ethanol (96–100%) to the lysate, and mix by vortexing.
    j. Carefully apply 600 μl lysate from step 9 to the QIAamp spin column. Close the cap and centrifuge for 1 min. Place the QIAamp spin column in a new 2 ml collection tube, and discard the tube containing the filtrate. Repeat step 10 until all of the lysate has been loaded on the column. (Close the QIAamp spin column before placing it in the microcentrifuge, taking care to avoid generating aerosols.
    k. Carefully open the QIAamp spin column and add 500 μl Buffer AW1. Centrifuge for 1 min. Place the QIAamp spin column in a new 2 ml collection tube, and discard the collection tube containing the filtrate.
    l. Carefully open the QIAamp spin column and add 500 μl Buffer AW2. Centrifuge for 3 min. Discard the collection tube containing the filtrate.
    m. Place the QIAamp spin column in a new 2 ml collection tube (not provided) and discard the old collection tube with the filtrate. Centrifuge for 3 min.
    n. Transfer the QIAamp spin column into a new, labeled 1.5 ml microcentrifuge tube (not provided) and pipet 50~100 μl Buffer ATE directly onto the QIAamp membrane. Incubate for 1 min at room temperature, then centrifuge for 1 min to elute DNA.

2. Sputum

  •  Notes before starting
    (1) Sample thawed to room temperature (15–25°C).
    (2) Equipment and Reagents to Be Supplied by User
    a. Suitable lab coat, disposable gloves, and protective goggles.
    b. Pipets (200μl,1000μl,10μl)
    c. Sterile pipet tips (pipet tips with aerosol barriers are recommended to help prevent cross-contamination)
    d. Thermoshaker at 56°C (capable of holding 2 ml collection tubes)
    e. Heating block or similar at 70°C (capable of holding 2 ml collection tubes)
    f. Microcentrifuge(capable of holding 2 ml collection tubes, the speed of centrifuge at 8000rpm or 14,000 rpm)
    g. Ethanol (96–100%)
    h. 2 ml microcentrifuge tubes
    (3)Kit Contents
    Kit Contents
    QIAamp UCP Pathogen Mini Kit
    Catalog no.
    Number of preps
    (50)
    50214
    50
    QIAamp UCP Mini Columns 50
    Collection Tubes(2ml) 50
    Tube Exlenders(3ml) 50
    Elution Tubes(1.5ml) 50
    Buffer ATL* 38ml
    Buffer APL2+ 14ml
    Buffer APW1+(concentrate) 18ml
    Buffer APW2+(concentrate) 15ml
    Buffer AVE* (concentrate) 8 vials
    Proteinase K 2.5ml
    Handbook 1
    * contains sodium azide as preservative
    + contains chaotropic salt. See page 7 for safety information
    (4)Preparation of buffers
    a. Buffer APW1:Before use, add 24 ml ethanol (96–100%) to 18 ml buffer APW1 concentrate to obtain 42 ml Buffer APW1. Mix well after adding ethanol.
    b. Buffer APW2:Before use, add 35 ml ethanol (96–100%) to 15 ml buffer APW2 concentrate to obtain 50 ml Buffer APW2. Mix well after adding ethanol.
    c. If a precipitate has formed in Buffer ATL or Buffer APL2, dissolve by incubating at 56°C.
    d. Equilibrate Buffer AVE to room temperature (15–25°C).
    e. Before use, add 100μl Reagent DX to 15 ml buffer ATL, Mix well after adding DX.
  •  Experimental steps
    (1) Mechanical Disruption
    Materials: Pathogen Lysis Tubes
    Procedures:
    a. Add 1.5 ml sputum to Pathogen Lysis tube, centrifuge at full speed (>14,000 x g) for 5 min.
    b. Discard the filtrate. Repeat step 1 and step 2 if essential. Note: Carefully pipet the filtrate, taking care to avoid pipet the beads.(vortex mixing samples are very important to ensure maximum concentration of DNA)
    c. Add 500 μl Buffer ATL (contain reg).
    d. Place the Pathogen Lysis Tube on a vortexer with the Microtube Foam Insert and vortex for 10 min at maximum speed.
    e. Remove the Pathogen Lysis Tube from the vortexer and centrifuge the tube for 5 s at 8,000 x g to remove drops from the inside of the lid.
    f. Transfer 400 μl supernatant from the Pathogen Lysis Tube into a fresh 2 ml microcentrifuge tube.
    (2) DNA Extraction (spin Protocol)(spin Protocol)
    This protocol is for purification of microbial DNA from 400 μl pretreated samples using a vacuum (recommended).
    Materials:QIAamp® UCP Pathogen Mini Kit
    Procedures:
    a. Add 40 μl Proteinase K and mix the sample by vortexing for 10 s.
    b. Incubate the sample at 56°C for 10 min.
    c. Add 200 μl of Buffer APL2 to the sample. Close the cap and mix by pulse-vortexing for 30 s.
    PS: In order to ensure efficient pathogen lysis, it is essential that the sample and Buffer APL2 are mixed thoroughly to yield a homogeneous solution.
    d. Incubate at 70°C for 10 min.
    e. Briefly spin the tube to remove drops from the inside of the lid.
    f. Add 300 μl ethanol to the lysate. Close the cap and mix thoroughly by pulse-vortexing for 15–30 s.
    g. Carefully apply 600 μl of the mixture from step 6 to the QIAamp UCP Mini spin column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate.
    PS:Close each spin column in order to avoid aerosol formation during centrifugation.
    h. Repeat step 7 by applying the remaining mixture from step 6 to the QIAamp UCP Mini spin column.
    i. Carefully open the QIAamp UCP Mini spin column and add 600 μl Buffer APW1 without wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp UCP Mini spin column in a clean 2 ml collection tube (not provided), and discard the collection tube containing the filtrate.
    j. Carefully open the QIAamp UCP Mini spin column and add 750 μl Buffer APW2 without wetting the rim. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min.
    k. Recommended: Place the QIAamp UCP Mini spin column in a new 2 ml collection tube (not provided) and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min.
    PS:This step helps to eliminate the chance of possible Buffer APW2 carryover.
    l. Place the QIAamp UCP Mini column into a new 2 ml collection tube. Open the lid and incubate the assembly at 56°C for 3 min to dry the membrane completely.
    m. Place the QIAamp UCP Mini column in a clean 1.5 ml elution tube and discard the collection tube. Carefully apply 20–100 μl of Buffer AVE to the center of the QIAamp UCP Mini membrane.
    Close the lid and incubate at room temperature for 1 min.
    Important:Important: Ensure that the elution buffer is equilibrated to room temperature. If elution is done in small volumes (<50 μl) the elution buffer has to be dispensed onto the center of the membrane for complete elution of bound DNA. Elution volume is flexible and can be adapted according to the requirements of downstream applications. The recovered eluate volume will up to 5 μl less than the elution volume applied onto the column.
    n. Centrifuge at full speed (20,000 x g; 14,000 rpm) for 1 min to elute the DNA.
    o. Repeat steps 13 and 14.

Ⅲ、454 Library Construction and Sequencing

1. PCR Amplification of 16s rDNA

(1) Primer: Choice the universal primer 341F and 805R of 16s rDNA V3-V5 variable region of. Amplification 460bp.
(2) PCR amplification
a. Dissolved primer 341F with 61μl DNase/RNase-Free Distilled Water to 100μM, then dilute with 1159μl DNase/RNase-Free Distilled Water to 5μM.
b. Dissolved primer 805R with 52μl DNase/RNase-Free Distilled Water to 100μM, then dilute with 988μl DNase/RNase-Free Distilled Water to 5μM.
c. PCR amplification reaction: Use the Q5 High-fidelity DNA polymerase of NEB, the total volume of each reaction is 50μl as following:
Q5 High-fidelity DNA polymerase 25μl
341Forward Primer(5μM) 5μl
805Reverse Primer(5μM) 5μl
DNA product 10μl
DNase/RNase-Free Distilled Water 5μl
d. Using the following thermal cycling profile for PCR
95℃    6min
30cycle:95℃ 40sec,58℃ 40sec,72℃ 60sec;
72℃    7min
4℃ Hold

2. PCR Product Purification

(1) Add 50μl AMPure XP beads to PCR tube, place the tube on a magnet to capture the beads. Incubate for 3-5min and discard the supernatant.
(2) Add 0.05% Tween20 to wash AMPure XP beads with twice time, place the tube on a magnet for 3-5min and discard the supernatant.
(3) Remove 100μl PCR product (add TE to 100μl if not enough) to the PCR tube from the step 2.
(4) Add 47μl , 2.5M NaCl to the tube. Incubate for 10min at room temperature. Place the tube on a magnet and incubate for 3-5min.
(5) Transfer 147μl supernatant to a new PCR tube, add 20μl AMPure XP beads and mix thoroughly.
(6) Incubate for 10min at room temperature. Place the tube on a magnet and incubate for 3-5min.
(7) Carefully remove and discard the supernatant.
(8) Add 200μl of 80% ethanol to wash AMPure XP beads. Repeat twice.
(9) Dry the beads at room temperature, until all of the ethanol has evaporated.
(10) Add 30μl DNase/RNase-Free Distilled Water to the tube and mix thoroughly.
(11) Place the EP tube on a magnet to capture the beads. Incubate for 3-5min.
(12) Carefully remove 30μl supernatant to a new EP tube.

3. PCR Product QC

(1) PCR product can be checked for concentration and purity by using NanoDrop 2000. The ratio of 1.8~2.0 is generally accepted as "pure" for DNA at 260/280nm. Nucleic acid concentration greater than 50ng/ul is accepted for the following experiment.
(2) Analyze each PCR product by using agarose gel electrophoresis.
(3)PicoGreen quantitative fluorescent QuantiFluor-ST fluorescence measurement standard and sample values, making the standard curve: y=0.2042x-0.0602, R - =0.9992. If the calculation of sample concentration, sample concentration higher than 20 ng/ L, you can continue to experiment.

Ⅳ、MiSeq Database building and computer operation

Roche 454 GS Junior Sequencing
(1) Library Construction
① End Repair Reaction Setup
a. Reaction Component
2.5 μl RL 10x Buffer
2.5 μl RL ATP
1 μl RL dNTP
1 μl RL T4 Polymerase
1 μl RL PNK
1 μl RL Taq Polymerase
9 μl Total volume
b. Reaction Program
25ºC for 20 min
72ºC for 20 min
4ºC on hold
② Adapter Ligation
a. Add 1μl RL Adaptor and 1μl RL Ligase to the tube
b. Incubate for 10min at 25ºC.
③ Remove the short fragment
a. Add Ampure beads to the tube from the step 2, mix thoroughly, incubate for 5min at room temperature.
b. Place the tube on a magnet to capture the beads. Incubate until the liquid is clear. Discard the supernatant.
c. Add 200μl of 80% ethanol to the tube and discard the supernatant. Repeat three times.
d. Dry the beads at room temperature, until all of the ethanol has evaporated.
e. Add 28ul Buffer-EB, mix using pipette tips carefully and incubate for 2min.
f. Place the tube on a magnet to capture the beads. Incubate until the liquid is clear. Remove 25ul supernatant to be stored at 4℃.
(2) Library quantification can be checked for concentration and purity by using NanoDrop 2000. The ratio of 1.8~2.0 is generally accepted as "pure" for DNA at 260/280nm. Nucleic acid concentration greater than 50ng/ul is accepted for the following experiment.
(3) Library quantification can be checked for the size of fragment by Agilent 2100 bioanalyzer. The peak value is generally accepted at 400-600bp.
(4) Performing a sequencing Run.