TITLE: DeRisi Lab RNA Library Prep 96-well Protocol  on Echo 550
AUTHORS: Madeline Y Mayday, Miriam R Simon, Lillian M Khan, Eric D. Chow, Matt S Zinter, Joseph Derisi

[DESCRIPTION]
Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS).  While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process.  Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. 

Here, we describe the integration of several instruments, including the Labcyte Echo 550 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries.

This protocol has been optimized for bronchoalveolar lavage fluid (BALF), nasopharyngeal swabs, RNA PAXgene blood, and plasma samples (see protocol variations for sample types needing RNA depletion).

[BEFORE_START]
Prep once:
A. Calibrating the vacuum concentrator. 
Test and calibrate all new plate brands or lot numbers by drying 5uL of water in each well to confirm plates dry appropriately (within 20-30 minutes).    
Temperature settings should be between 40-45 °C (could vary slightly depending on model).
Test for minimum drying time.

B. Calibrating all plates for liquid handlers. 
It is vital to input and test labware definitions of all plates and confirm all liquid handlers have appropriate liquid transfer settings.

C. Prep ERCCs.
External RNA Control Consortium (ERCC) RNA Spike-In Mix (Cat No. 4456740) was used in this study as a control for quality of sequencing libraries. Stock concentrations were quantified using Qubit RNA HS assay and diluted in nuclease free water to a target stock concentration of 50pg/uL.  This stock was divided into small aliquots and stored at -80ºC for one time use.

Prep every time:
A. Prep master mix calculations and sample sheets.
In order to program the Echo dispense protocols to reflect the right transfer volumes and destination wells, it is helpful to prepare a sample sheet listing the well location of each sample.

Prep before PCR amplification step:
A. Barcode plate.
Prepare or obtain a 96-well PCR plate with at least 10uL of 5uM unique dual indexing primers in each necessary well.  This plate must be prepared before the post-ligation bead clean as it is used for the elution step.
B. USER/PCR plate.
Using the Echo, prepare a 96-well PCR plate with 10.9uL of USER/PCR master mix in each necessary well.  This plate must be prepared before the post-ligation bead clean as it is used as the final plate.

[GUIDELINES]
Pre-PCR BSL-1 Guidelines
Absolutely no amplicons allowed in this room, thermocyclers should NOT be used for PCR
Although a lab coat is not necessary for all BSL-1 work, always wear a lab coat and gloves when doing library prep or working with patient samples and any isolated nucleic acid prior to the final amplification stage
Wipe down workspace with 70% ethanol and RNAse-ZAP before commensing work
Clean up lab space after work is complete

Protocol Specific Guidelines
Prepare master mix reagents in a hood the day of use to preserve efficiency of all enzymes. 
Only use consumables and pipettes that have are designated for hood use.
Take Ampure beads out of 4C and room temp 30 minutes before beads cleans.

Equipment Guidelines 
Vacuum Evaporators
Different model vacuum evaporators vary in sample dehydration times. To confirm the minimal drying time for each instrument, fill each well of a 96-well PCR plate with 5uL water and spin at 37ºC-38ºC until completely dry. 
Variations in the manufacturing of different brands of PCR plates may also inhibit efficient sample drying. To prevent excessive drying times and potential compromise of RNA, test the desired brand of PCR plate by filling each well with 5uL water and ensuring it dries within 20-30 minutes.  Over-drying and over-heating may cause degradation of the RNA.
 
Labcyte Echo 525 
When calculating master mix volumes, be sure to make a sufficient amount of each reagent to account for the minimum and maximum working volume of source plates.
384 PP Plus Echo Qualified source well working volume: 20 - 65uL (dead volume of ~20uL)

[STEPS]
2. Spin plate of sample RNA in vacuum evaporator at the appropriate temperature and time settings to dry completely (approximately 37-38ºC for 1 hour, depending on number of samples and machine used).
 
SECTION: GeneVac
1. Warming up GeneVac: Turn to "aqueous" setting and set temperature to ~40-42ºC and let run for ~30min before use.
SECTION: PREPARE MASTER MIX REAGENTS (DAY 1)
3. Prepare enough fragmentation, first strand, and second strand master mixes for each sample. For whole blood, plasma or other samples requiring rRNA/globin RNA depletion, see fragmentation modifications below. 
 


Be sure to take into account the dead volume and the maximum working volume of the source plate being used. For example, in a 384-well Echo-qualified source plate, the volume of reagent in each well must be between 20uL and 65uL to ensure accurate dispensing.


 
 
 
 
SECTION: PREPARE MASTER MIX REAGENTS (DAY 1)
5. Spin the Echo source plate in the centrifuge for 5 minutes at 2000 rpm to rid source wells of any bubbles produced in manual pipette transfer of master mixes. Reagents should be at room temp before Echo transfer.

SECTION: DISPENSING FRAGMENTATION REAGENTS
6. For 384-well source plates, use the Echo's “Survey” setting to determine volume in each source well to ensure volume is 20<x<65uL to account for the minimum and maximum working volume.

SECTION: DISPENSING FRAGMENTATION REAGENTS
7. Using the BP setting, dispense 1000nL of fragmentation master mix (1100nL for FastSelect) into each sample well.
 

SECTION: DISPENSING FRAGMENTATION REAGENTS
8. Remove sample plate and seal with a foil seal.  Gently vortex sample plate to mix, followed by quick spin.
SECTION: FRAGMENTATION INCUBATION (for FastSelect only)
9. This step is ONLY for samples with FastSelect depletion. For all other sample types, proceed to Step 10.
 
 FRAGMENTATION INCUBATION (heated lid set to 105ºC)•  1 minutes at 94°C•  2 minutes at 75°C•  2 minutes at 70C•  2 minutes at 65°C•  2 minutes at 60°C•  2 minutes at 55°C•  5 minutes at 37°C•  5 minutes at 25°C
 

SECTION: DISPENSING FIRST STRAND SYNTHESIS REAGENTS
10. Remove foil seal, and load into Echo.  Using the GP setting, dispense 1000nL of first strand synthesis master mix into each sample well needed for reaction.

SECTION: DISPENSING FIRST STRAND SYNTHESIS REAGENTS
11. Remove sample plate and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
SECTION: FIRST STRAND SYNTHESIS INCUBATION
12.  
First Strand Synthesis IncubationHeated Lid: 105 °CTemperature (°C)Time (mins)2510421570154Hold
 
    
SECTION: DISPENSING SECOND STRAND SYNTHESIS REAGENTS
13. Remove sample plate from thermocycler, remove foil seal, and load into Echo.  Using the GP setting, dispense 6000nL second strand synthesis master mix into each sample well.

SECTION: DISPENSING SECOND STRAND SYNTHESIS REAGENTS
14. Remove sample plate and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
SECTION: SECOND STRAND SYNTHESIS INCUBATION
15.  
SECTION: NUCLEIC ACID PURIFICATION: HAND BEAD CLEAN
16. Bead clean using a 1.4x Ampure bead-to-sample ratio. If you make your own SPRI beads, you will need to adjust the ratio to match the 1.4x Ampure bead 
 
 
 Prepare 80% ethanol (fresh, 50 mL reservoir) and beads (50 mL reservoir, left out at RT for 30 min prior to use)•  add 12 uL H2O to sample wells to bring up to 20 uL•  Add 28 uL beads to sample plate, mix, and incubate 5 minutes (visually confirm sufficient mixing)•  Move sample plate to magnet and incubate 5 minutes•  Remove 48 uL supernatant and transfer to waste•  Wash with 150uL 80% ethanol, pause 30 seconds, remove and move to waste•  Repeat ethanol wash step•  Air dry beads for 10 minutes *do not overdry*•  Move sample plate off magnet•  Resuspend beads in 6uL eluent (water), mix, and incubate 5 minutes (visually confirm sufficient mixing)•  Move sample plate to magnet and incubate 3 minutes•  Aspirate 5uL of supernatant (containing cDNA) and transfer into a new clean PCR plate•  Seal final plate and place in -20 until Day Two
 
 
 


SECTION: PREPARE MASTER MIX REAGENTS (DAY 2)
17. Prepare enough end prep, adaptor ligation, USER/PCR master mixes, and adaptor dilutions for each sample.
 
Be sure to take into account the dead volume and the maximum working volume of the source plate. For example, in a 384-well Echo source plate, the volume of reagent in each well must be between 20uL and 65uL to ensure accurate dispensing. USER/PCR mix is very viscous and easily bubbles, do not go to second stop when dispensing into 6-Res Echo source plate.
 

 

 
 
 
 
 
SECTION: PREPARE MASTER MIX REAGENTS (DAY 2)
19. Spin the Echo source plate in the centrifuge for 5 minutes at 2000 rpm to rid source wells of any bubbles produced in pipette transfer of master mixes.

SECTION: DISPENSING END PREP REAGENTS
20. For 384-well source plates, use the Echo's “Survey” setting to determine volume in each source well to ensure volume is 20<x<65uL to account for the minimum and maximum working volume.
SECTION: DISPENSING END PREP REAGENTS
21. Using the BP setting, dispense 1000nL of end prep master mix into each sample well.
 

SECTION: DISPENSING END PREP REAGENTS
22. Remove sample plate and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
SECTION: END PREP INCUBATION
23.  
End Prep IncubationHeated Lid: >75 °CTemperature (°C)Time (mins)2030653010Hold
 
    

SECTION: DISPENSING ADAPTOR LIGATION REAGENTS
24. Remove sample plate from thermocycler, remove foil seal, and load into Echo.  Using the GP setting, proceed with 2 transfers. First, transfer 3100nL of adaptor ligation master mix. Then, transfer 250nL of appropriately diluted adaptor to each sample well. Make sure to add these reagents separately; this will prevent excessive adaptor dimer. 

SECTION: DISPENSING ADAPTOR LIGATION REAGENTS
25. Remove sample plate and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
SECTION: ADAPTOR LIGATION INCUBATION
26.  
Adaptor LigationHeated Lid: OffTemperature (°C)Time (mins)201510Hold
 
    

SECTION: USER/PCR SETUP - REAGENT PLATE
27. Before bead cleaning the sample post-adaptor ligation, prepare the USER/PCR master mix plate.  It will be used as the final destination of cDNA during post-adaptor ligation bead clean.
 
 
 

SECTION: NUCLEIC ACID PURIFICATION: HAND BEAD CLEAN
29. Take beads out of 4C 30 min before bead cleaning to bring to room temp. Bead clean the end-prepped, adaptor-ligated cDNA using a sample to Ampure bead solution ratio of 0.8x.
 
 
 Prepare 80% ethanol (fresh, 50 mL reservoir) and beads (50 mL reservoir)•  Add 25.65 uL H2O to sample wells to bring up to 35 uL•  Add 28 uL beads to sample plate, mix, and incubate for 5 minutes (visually confirm sufficient mixing)•  Move sample plate to magnet and incubate 5 minutes•  Remove 64 uL supernatant and transfer to waste•  Wash with 150uL 80% ethanol, pause 30 seconds, remove and move to waste•  Repeat ethanol wash step•  Air dry beads for 10 minutes *do not overdry*•  Move sample plate off magnet•  Resuspend beads in 6.5uL eluent (barcodes), mix, and incubate 3 minutes (visually confirm sufficient mixing)•  Move sample plate to magnet and incubate 5 minutes•  Aspirate 5uL of supernatant (containing adaptor-ligated cDNA and barcodes) and transfer into the final plate prepared with USER/PCR master mix•  Gently vortex and quick-spin plate to mix
 

 


SECTION: NUCLEIC ACID PURIFICATION: HAND BEAD CLEAN
31. Bead clean the post USER/PCR cDNA on the Biomek using a sample to Ampure bead solution ratio of 0.8x.
 
Use Nuclease-free water to elute sample from beads.
 
 
 *The following bead clean should be done on the bench or anywhere BUT the pre-PCR hood Prepare 80% ethanol (fresh, 50 mL reservoir) and beads (50 mL reservoir)•  Add 12.72 uL beads to sample plate, mix, and incubate 5 minutes (visually confirm sufficient mixing)•  Move sample plate to magnet and incubate 5 minutes•  Remove 27.5 uL supernatant and transfer to waste•  Wash with 150uL 80% ethanol, pause 30 seconds, remove and move to waste•  Repeat ethanol wash step•  Air dry beads for 10 minutes *do not overdry*•  Move sample plate off magnet•  Resuspend beads in 33uL eluent (water), mix, and incubate 3 minutes (visually confirm sufficient mixing)•  Move sample plate to magnet and incubate 5 minutes•  Aspirate 31uL of supernatant (containing cDNA) and transfer into a new clean Echo source plate •  Seal final plate and freeze for downstream use - using an Echo plate for the final plate means pooling can be done with the Echo•  use a regular 384PP source plate rather than an LDV plate - LDV plates seem to bind DNA and cause loss of library•  Remaining ~1uL in the bead plate can be recovered and used for QuBit (to test several samples for successful library prep)
 


 

SECTION: PREPARE MASTER MIX REAGENTS (DAY 1)
4. Load reagents into a 384PP Plus Echo source plate by pipetting reagents into each required well and seal with a foil seal.  Pipet carefully to avoid formation of bubbles. Ensure that wells with master mix are noted to make Echo CSV with transfer specifications for each well.




SECTION: PREPARE MASTER MIX REAGENTS (DAY 2)
18. Load reagents into Echo source plates by pipetting reagents into each required well and seal with a foil seal.  Pipet carefully to avoid formation of bubbles.


 
SECTION: FINISHED LIBRARIES
32. Finished libraries are now ready to be quality checked, and/or undergo equal volume pooling to determine representation of each library in the pool.  This can be used to determine the volume of each sample needed for equal pooling.
 
SECTION: USER/PCR SETUP - REAGENT PLATE
28. Load a new, sterile, empty PCR plate into the Echo destination port.  Using the GP setting, dispense 10,900nL of USER/PCR master mix from a 6-Res plate into each sample well.

SECTION: USER/PCR INCUBATION
30.  
   USER/PCR INCUBATION (heated lid set to 105oC) cycle #37ºC for 15 mins198ºC for 30s198ºC for 10s12-18**NOTE: number of PCR cycles should be chosen based on approximate RNA sample input65ºC for 75s PAXgene and NP: 1465ºC for 5mins1BAL: 16-18Hold at 4ºC∞