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.
• When starting the protocol, turn on the computer first, THEN Rosie.  When finished, turn Rosie off first, followed by the computer.
• 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 (BMC Final F, BMC Final R)
• HF Phusion (NEB, M0530L)
• KAPA SYBR 2xMM for final QPCR (can be purchased from the BMC)
• Invitrogen Super magnet (16 or 8 sample capacity)
• 96 well magnet plate
• up to 10 boxes of 300uL filtered epTIPS (Eppendorf, Cat# 0030014456, reloads Cat# 0030014472)
• up to 13 boxes of 300uL unfiltered epTIPS (Eppendorf, bulk Cat# 960050620)
• 8 boxes of 50uL filtered epTIPS (Eppendorf, Cat# 0030014413, reloads Cat# 0030014430)

• up to 10 30mL reagent reservoirs (Eppendorf, 250 reservoirs Cat# 960051500)  
• up to 2 100mL reagent reservoirs (Eppendorf, 250 reservoirs Cat# 960051511)

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 prompt half way through the protocol that will ask if you are running two plates)

Materials used:

• Contents of MM
• 384 well Lightcycler 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 Lightcycler 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 (Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls)
Breakdown of QPCR amplification math (done to normalize each sample):
○   delta Ct = Sample Ct - highest Ct in sample set
○   fold = 1.75^(delta Ct)
○   dilution needed = fold
○  note - that input is 2uL per RXN so sample with highest 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 using the Initial Dilution Sample and H2O pages from the Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls saved as two individual CSV files and imported to Rosie.

- please run the H2O protocol first

Materials used:

• 50uL filtered epTIPS x 2
• 30mL reagent reservoir x 1
• EB
• 1 Axygen 96 well skirted PCR plate

Library Preparation:

Step 1 (one plate at a time)

Rosie Protocol Used: '16s Step 1 PCR.dws'

Materials used:

• 4 Axygen 96 well skirted PCR plates
• 1 row of a 300uL non-filtered epTIPS box
• 1 box of 50uL filtered epTIPS
• 1 30mL reagent reservoir
• Clear PCR plate covers

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 Cycling 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:

Rosie Protocol Used: '4x to 96pool.dws'

Materials used:

• 1 Axygen 96 well skirted PCR plate
• 1 box of 300uL filtered epTIPS

96 well SPRI Clean Up

Rosie Protocol Used: 'SingleSPRI.dws'

Materials used:

• SPRI beads (9mL)
• 70% EtOH (32mL)
• EB (4.5mL)
• 96 well magnet plate
• 3 30mL reagent reservoirs
• 1 100mL reagent reservoir (for waste)
• 1 Axygen 96 well skirted PCR plate
• 3 boxes of 300uL filtered epTIPS
• 5 boxes of 300uL un-filtered epTIPS

Do not put cover on EtOH reservoir tightly after filling, the volume will be too high to do this with out spilling.

Note: At one point during the protocol, Rosie will ask the user to replace empty tip boxes.  Replace the boxes in B1 and B2 with new boxes.  When the protocol is restarted, it will prompt you to take action on the empty tip box in B4.  Choose “ignore” and the program will continue without problems.

Step 2 (one plate at a time):

Rosie Protocol Used: '16s Step2 PCR.dws'

Materials used:

• 4 Axygen 96 well skirted PCR plates
• 1 pre-pipetted PE-PCR-IV-XXX barcode plate
• 3 boxes 50uL filtered epTIPS
• 1 30mL reagent reservoir
• Clear PCR plate covers

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

16s Step 2 Cycling 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:

Rosie Protocol Used: '4x to 96pool.dws'

Materials used:

• 1 Axygen 96 well skirted PCR plate
• 1 box of 300uL filtered epTIPS

** Now you have a choice!***

#1 - If your sample plate is a mixture of sample types (stool and saliva for example) or you have reason to suspect something went wrong

#2 - If your sample plate is all the same sample type all samples can be pooled 1:1 after

Choice #1:

96 well SPRI Clean Up

Rosie Protocol Used: 'SingleSPRI.dws'

Materials used:

• SPRI beads (9mL)
• 70% EtOH (32mL)
• EB (4.5mL)
• 96 well magnet plate
• 3 30mL reagent reservoirs
• 1 100mL reagent reservoir (for waste)
• 1 Axygen 96 well skirted PCR plate
• 3 boxes of 300uL filtered epTIPS
• 5 boxes of 300uL un-filtered epTIPS

Do not put cover on EtOH reservoir tightly after filling, the volume will be too high to do this with out spilling.

Note: At one point during the protocol, Rosie will ask the user to replace empty tip boxes.  Replace the boxes in B1 and B2 with new boxes.  When the protocol is restarted, it will prompt you to take action on the empty tip box in B4.  Choose “ignore” and the program will continue without problems.

Final QPCR (this is a copy of the BMC QPCR for quality control, it can be modified to use normal SYBR green as well):
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 Lightcycler)

Activation:
95°C - 5 minutes
Amplification:
95°C – 30 seconds
60°C – 45 seconds

Run 35 cycles of amplification

Use mid-log phase of curves to determine volumes for multiplexing (use Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls)
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 - highest 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 highest Ct will get an undiluted XuLs added to final multiplex, X can be raised or lowered to accommodate the needed volume of other samples

Multiplexing:

Rosie protocol used: '96 to 1 pool_postSPRI.dws'

Materials used:

• 2 1.5mL eppendorf tubes (one for samples and one for negatives)
• 1 box of 300uL filtered epTIPs

- The above protocol imports volume information using the Final Multiplex page from the Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls saved as a CSV files and imported to Rosie

- Please note that the tube rack takes up two deck positions, this is not shown on Rosie's deck display

Choice 2:

Multiplexing:

Rosie protocol used: '96 to 1 pool_preSPRI.dws'

Materials used:

• 2 1.5mL eppendorf tubes (one for samples and one for negatives)
• 1 box of 300uL filtered epTIPs

- The above protocol imports volume information using the Final Multiplex page from the Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls saved as a CSV files and imported to Rosie

- Please note that the tube rack takes up two deck positions, this is not shown on Rosie's deck display

SPRI (for individual tubes of pooled samples):

Materials used:

• SPRI beads (85% of total sample volume)
• 70% EtOH (2mLs per sample tube)
• EB (200uL per sample tube)
• Invitrogen Super magnet (16 or 8 sample capacity)

- allow SPRI beads to warm to RT

- Aliquot SPRI beads into tubes containing sample mixture (SPRI volume should be 85% of sample volume)

- Vortex well and incubate for 13 minutes at RT

- Separate on magnet for 2 minutes

- While on magnet, remove/discard SN

- Wash beads 2x with 1mL of 70% EtOH

- Air dry beads for 15-20 minutes on magnet

- remove tubes from magnet, add 200uL of EB to tubes

- vortex well, incubate for 7 minutes at RT (still off the magnet)

- Separate on magnet for 2 minutes

- transfer SN to new tube

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 Lightcycler)

Activation:
95°C - 5 minutes
Amplification:
95°C – 30 seconds
60°C – 45 seconds

Run 35 cycles of amplification

Use mid-log phase of curves to determine volumes for multiplexing (use Illumina Library Construction/Tools/Attachments/16s primer_amp_QPCR sheets.xls)
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 - highest 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 highest 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:
- Run a Bioanalyzer DNA HighSensitivity on all sample poolings as well as final multiplexed lane to confirm library size (~450bp) and concentration

Please note - a peak at ~120bp is usually primer dimers, if this peak is large you will need to repeat the SPRI clean up to remove it or will need to gel purify your sample. 

- Aliquot ~20uL of the final mix and submit for sequencing