...
- 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.
...
- 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 BMC Final F, PE Seq 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
- X boxes of 300uL filtered epTIPS (Cat#XX)
- X boxes of 300uL unfiltered epTIPS (Cat#XX)
- X boxes of 50uL filtere epTIPS (Cat#XX)
...
- 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
...
** 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 Program (Opticon or Lichtcycler)
HeatActivation:
95°C – - 5 minutes
Amplify Amplification:
95°C – 10 30 seconds
60°C – 20 seconds45 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 (use Illumina Library QPCR and Multiplexing documentConstruction/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 tubeSPRI Clean Up
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)
HeatActivation:
95°C – - 5 minutes
AmplifyAmplification:
95°C – 10 30 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 45 seconds
Run 35 cycles of amplification
Use mid-log phase of curves to determine volumes for multiplexing (google docs, Illumina Library QPCR and Multiplexinguse 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 Once samples have been multiplexed aliquot ~20uL of the final mix and submit it to the BioMicro Center for sequencing