16S Library Multiplex Preparation (Automated) - in progress

16S Library Multiplex Preparation (Automated) 

Note: 

• This protocol is for preparing 16S libraries on Rosie (the Alm lab's epMotion 5075)  and should only be used to prepare 96 well sample plates for multiplexing. 
• At the beginning of EACH DAY using Rosie spray and wipe down the deck with RNAse AWAY and 70% EtOH then UV the deck for 10-15minutes before starting protocols for the day. 
• If you are preparing only one plate for sequencing please use the PE_16s_V4_U515_F primer in the first step PCR. If you are planning on combining multiple 96 well plates for sequencing (4 or more recommended) please use the PE_16s_V4_F_Bar## primer for your first step PCR. 

Materials:

• Agencourt Ampure XP, A63881 (60mL, $300)
• 2 Roche LichtCycler480 384-well plate 
• 1:100 dilution of SYBR stock (Invitrogen S7563, 10,000x)
• Step 1/ Initial QPCR Primers ( PE_16s_v4U515-F)
• Step 2 primers ( PE-III-PCR-F, PE-IV-PCR-XXX; NOTE - PE-IV-PCR-XXX primers should be used as a pre-pipetted 96well plate)
• Final QPCR primers (PE Seq F, PE Seq R)
• HF Phusion (NEB, M0530L)
• KAPA SYBR 2xMM for final QPCR
• Invitrogen Super magnet (16 or 8 sample capacity)
• X boxes of 300uL filtered epTIPS (Cat#XX)
• X boxes of 300uL unfiltered epTIPS (Cat#XX)
• X boxes of 50uL filtere epTIPS (Cat#XX)

Determination of  Step 1 Cycle Time and Sample Check:

Rosie Protocol Used: '16s Stp1 384 QPCR.dws', please note that this protocol can be run for 1 or 2 sample plates at once (there is a promt half way through the protocol that will ask if you are running tow plates)

Materials used:

• Contents of MM
• 384 well Lichtcycler QPCR plate
• Clear PCR plate covers
• 50uL filtered epTIPS x 2
• 300uL non-filtered epTIPS x 1
• 30mL reagent reservoir x 1

Run this step in duplicate or triplicate to best estimate proper cycling time

Initial QPCR Program (Roche Lighcycler 480):
Heat:
98°C – 30 seconds
Amplify:
98°C – 30 seconds
52°C – 30 seconds
72°C – 30 seconds
Cool:
4°C - continuous
Use Ct (bottom of curve, not mid-log) of curves to determine dilutions for step 1 amplification (Please reference Illumina Library QPCR and Multiplexing document)
Breakdown of QPCR amplification math (done to normalize each sample):
○   delta Ct = Sample Ct - lowest Ct in sample set
○   fold = 1.75^(delta Ct)
○   dilution needed = fold
○  note - that input is 2uL per RXN so sample with lowest Ct gets 2uL undiluted
Please note – samples may fail due to too little, too much material, or a poor reaction. It is recommended that failed samples be re-run before moving forward

Sample Normalization (one plate at a time):

Rosie Protocol Used: 'Initial Dilu-Sample2.dws' and 'Initial Dilution-H2O.dws'

- The above protocols import volume information from CSV files (please see XXX files for this purpose)

- please run the H2O protocol first

Materials used:

• 50uL filtered epTIPS x 2
• 30mL reagent reservoir x 1
• EB

Library Preparation:
Step 1 (one plate at a time)

Please Note: Samples are run as four 25uL reactions that are pooled at end of cycling

1st step Master Mix 25uL RXN (MM1)

16SStep 1 Program:
Heat:
98°C – 30 seconds
Amplify:
98°C – 30 seconds
52°C – 30 seconds
72°C – 30 seconds
Cool:
4°C - continuous
Run amplification cycle number determined via QPCR (no more than 20 cycles allowed)
After cycling pool duplicates, now have 1x 100uL reaction per sample

SPRI Clean Up
Materials used:
○   SPRI beads
○   70% EtOH
○   EB
○   Invitrogen super magnet
- Vortex cold AmpureXP beads, pool DNA from PCR tubes (~100uL)
- Aliquot 85.5uL beads into Epi’s – let equilibrate to RT
- Add DNA (take 95uL) + beads (85.5uL) = 180.5 uL
- Incubate 13 mins @ RT
- Separate ON magnet 2 mins
- While ON magnet, remove/discard SN
- Wash beads 2x with 70% EtOH, 500uL each wash
- Air dry beads for 15-20mins on magnet
- Remove from magnet, elute in 40uL H2O, vortex to resuspend
- Incubate 7 mins @ RT
-  Separate on magnet 2mins
- Collect 35-40 ul and save SN

Sample Re-Aliquoting and Step 2
Please Note: Samples are run as four 25uL reactions that are pooled at end of cycling

16s Step 2 Program:

Heat:
98°C – 30 seconds
Amplify:
98°C – 30 seconds
83°C – 30 seconds
72°C – 30 seconds
Cool:
4°C - continuous
Run 7 cycles of amplification
- After cycling pool duplicates, now have 1x 100uL reaction per sample

SPRI Clean Up
Materials used:
○   SPRI beads
○   70% EtOH
○   EB
○   Invitrogen super magnet
- Vortex cold AmpureXP beads, pool DNA from PCR tubes (~100uL)
- Aliquot 85.5uL beads into Epi’s – let equilibrate to RT
- Add DNA (take 95uL) + beads (85.5uL) = 180.5 uL
- Incubate 13mins @ RT
- Separate ON magnet 2mins
- While ON magnet, remove/discard SN
- Wash beads 2x with 70% EtOH, 500uL each wash
- Air dry beads for 15-20mins on magnet
- Remove from magnet, elute in 40uL H2O, vortex to resuspend
- Incubate 7mins @ RT
-  Separate on magnet 2mins
- Collect 35-40 ul and save SN

Final QPCR
Once you have a substantial or all of your samples prepared you can run a final QPCR to determine dilutions and volumes for multiplexing. This step also confirms that the library preparation was successful

Final QPCR Program (Opticon or Lichtcycler)

Heat:
95°C – 5 minutes
Amplify:
95°C – 10 seconds
60°C – 20 seconds
72°C – 30 seconds
Melting Curve:
95°C – 5 seconds
65°C – 1 minute
97°C - continuous
Cool:
40°C – 10 seconds
Run 35 cycles of amplification
Use mid-log phase of curves to determine volumes for multiplexing (google docs, Illumina Library QPCR and Multiplexing)
Please note – samples may fail due to too little, too much material, or a poor reaction. It is recommended that failed samples be re-run before moving forward
Breakdown of QPCR multiplexing math (done to normalize each sample):
○  delta Ct = Sample Ct - lowest Ct in sample set
○  fold = 1.75^(delta Ct)
○  ratio = 1/fold
○  volume to mix b/c of ratio = X*ratio (X = minimum desired volume per sample)
○  how to dilute = fold
○  note - sample with lowest Ct will get an undiluted Xuls added to final multiplex, X can be raised or lowered to accommodate the needed volume of other samples

Sample Multiplexing and Submission for Sequencing:
- Once samples have been multiplexed aliquot ~20uL of the final mix and submit it to the BioMicro Center for sequencing