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 (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 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 (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 - 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 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 (8.5mL)
• 70% EtOH (30.2ml)
• EB (4.1mL)
• 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

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 (8.5mL)
• 70% EtOH (30.2ml)
• EB (4.1mL)
• 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

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 Lichtcycler)

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 - 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

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 Lichtcycler)

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 - 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:
- 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