TITLE: CZ Biohub RNA Library Prep Protocol on Echo 550
AUTHORS: Saharai Caldera, Madeline Mayday, Lillian Khan, Eric D. Chow, Matt S. Zinter, Joseph L. DeRisi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = ":justify;">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. </span></div><div class = "text-block"><span style = ":justify;">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.  For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.</span></div></div>

[STEPS]
?. Spin plate of sample RNA in vacuum evaporator at the appropriate temperature and time settings to dry completely (approximately 40ºC for 25-30 minutes, depending on number of samples and machine used).
?. [PREPARE INITIAL RNA SAMPLE PLATE]
Load 5uL of sample RNA into a 384-well PCR plate. Repeat with all desired samples.  Record well location of each sample.
It is convenient to organize the 384-well plate into 4 quadrants of 96 samples. This will allow for multichannel dispensing of samples using a pipette, providing an easy transition between manual and automated steps.
?. [PREPARE MASTER MIX REAGENTS]
Prepare enough fragmentation, first strand, and second strand master mixes for each sample.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.  AB1Fragmentation Master Mix (1x)2Reagent (lilac&nbsp;tubes)Volume (uL per sample)3ERCC (stock concentration 50pg/uL)0.54First Strand Synthesis Reaction Buffer0.45Random Primers0.16Total Reaction Volume1.07&nbsp;8First Strand Synthesis Master Mix (1x)9Reagent (lilac tubes)Volume (uL per sample)10Fragmentation Reaction Volume1.011NEBNext First Strand Synthesis Enzyme Mix0.212Nuclease Free Water0.813Total Reaction Volume
2.014&nbsp;15Second Strand Synthesis Master Mix (1x)16Reagent (orange tubes)Volume (uL per sample)17First Strand Reaction Volume2.018Second Strand Synthesis Reaction Buffer0.819Second Strand Synthesis Enzyme Mix0.420Nuclease Free Water4.821Total Reaction Volume8.0
AB1Fragmentation Master Mix (1x)2Reagent (lilac&nbsp;tubes)Volume (uL per sample)3ERCC (stock concentration 50pg/uL)0.54First Strand Synthesis Reaction Buffer0.45Random Primers0.16Total Reaction Volume1.07&nbsp;8First Strand Synthesis Master Mix (1x)9Reagent (lilac tubes)Volume (uL per sample)10Fragmentation Reaction Volume1.011NEBNext First Strand Synthesis Enzyme Mix0.212Nuclease Free Water0.813Total Reaction Volume
2.014&nbsp;15Second Strand Synthesis Master Mix (1x)16Reagent (orange tubes)Volume (uL per sample)17First Strand Reaction Volume2.018Second Strand Synthesis Reaction Buffer0.819Second Strand Synthesis Enzyme Mix0.420Nuclease Free Water4.821Total Reaction Volume8.0
?. [PREPARE MASTER MIX REAGENTS]
Spin the Echo source plate in the centrifuge for 5 minutes at 1500 rcf to rid source wells of any bubbles produced in manual pipette transfer of master mixes.
Time and rpm was per recommendation by Howard Lee at Labcyte.Reagents must come to room temperature before dispensing on the Echo.
?. [DISPENSING FRAGMENTATION REAGENTS]
For 384-well source plates, use the Echo's “Survey” setting to determine volume in each source well to ensure volume is 20
?. [DISPENSING FRAGMENTATION REAGENTS]
Using the BP setting, dispense 1000nL of fragmentation master mix into each sample well.
Ensure there are no bubbles present in the source plates after spinning.
?. [DISPENSING FRAGMENTATION REAGENTS]
Remove sample plate and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
?. [FRAGMENTATION INCUBATION]
AB1Fragmentation Incubation2Heated Lid: 105 °C3Temperature (°C)Time (mins)4 941254Hold
AB1Fragmentation Incubation2Heated Lid: 105 °C3Temperature (°C)Time (mins)4 941254Hold
?. [DISPENSING FIRST STRAND SYNTHESIS REAGENTS ]
Remove sample plate from thermocycler, remove foil seal, and load into Echo.  Using the BP setting, dispense 1000nL of first strand synthesis master mix into each sample well needed for reaction.
?. [DISPENSING FIRST STRAND SYNTHESIS REAGENTS ]
Remove sample plate and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
?. [FIRST STRAND SYNTHESIS INCUB]
AB1First Strand Synthesis Incubation2Heated Lid: 105 °C3Temperature (°C)Time (mins)42510542156701574Hold  AB1First Strand Synthesis Incubation 
2Heated Lid: 105 °C
3Temperature (°C)
425
542
670
74
AB1First Strand Synthesis Incubation2Heated Lid: 105 °C3Temperature (°C)Time (mins)42510542156701574Hold
AB1First Strand Synthesis Incubation 
2Heated Lid: 105 °C
3Temperature (°C)
425
542
670
74
?. [DISPENSING SECOND STRAND SYNTHESIS REAGENTS]
Remove sample plate from thermocycler, remove foil seal, and load into Echo.  Using the BP2 setting, dispense 6000nL second strand synthesis master mix into each sample well.
?. [DISPENSING SECOND STRAND SYNTHESIS REAGENTS]
Remove sample plate and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
?. [SECOND STRAND SYNTHESIS INCUBATION]
AB1Second Strand Synthesis Incubation 
2Heated Lid: Off
3Temperature (°C)
Time (mins)416
60510Hold
AB1Second Strand Synthesis Incubation 
2Heated Lid: Off
3Temperature (°C)
Time (mins)416
60510Hold
?. [NUCLEIC ACID PURIFICATION: BEAD CLEAN]
Bead clean on Biomek 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 Use nuclease free water to elute sample from beads.   AB1Post Second Strand Bead Clean (1.4x)2Bead Clean StepsVolumes per sample(uL)3Initial Sample Volume8.04Ampure Bead Volume11.25Elution Volume6.06Recover Volume5.0
AB1Post Second Strand Bead Clean (1.4x)2Bead Clean StepsVolumes per sample(uL)3Initial Sample Volume8.04Ampure Bead Volume11.25Elution Volume6.06Recover Volume5.0
?. [PREPARE MASTER MIX REAGENTS]
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.  AB1End Prep Master Mix (1x)2Reagent(green tubes)Volume per sample(uL)3Post-Second Strand Synthsis Bead Clean Volume5.04Ultra II End Prep Reaction Buffer0.75Ultra II End Prep Enzyme Mix0.36Total Volume6.07&nbsp;&nbsp;8Adaptor Ligation Master Mix (1x)9Reagent(red tubes)Volume per sample(uL)10End Prep Reaciton Volume6.011NEBNext Ultra II Ligation Master Mix3.012NEBNext Ligation Enhancer0.113Total Volume9.114&nbsp;&nbsp;15Adaptor Master Mix (1x)16Reagent(red tube)Volume per sample(uL)17Diluted Adaptor0.2518Note: Adaptor should be diluted based on approximate sample input and should not be added to adaptor ligation master mix to avoid adaptor-dimers.&nbsp;19&nbsp;&nbsp;20USER/PCR Master Mix (1x)21Reagent(USER- white tube; Q5- blue tube)Volume per sample(uL)22Adaptor Ligation Reaction Volume5.023Nuclease Free Water2.524NEB USER Enzyme0.925NEBNext Ultra II Q5 Master Mix7.526Total Volume15.9
AB1End Prep Master Mix (1x)2Reagent(green tubes)Volume per sample(uL)3Post-Second Strand Synthsis Bead Clean Volume5.04Ultra II End Prep Reaction Buffer0.75Ultra II End Prep Enzyme Mix0.36Total Volume6.07&nbsp;&nbsp;8Adaptor Ligation Master Mix (1x)9Reagent(red tubes)Volume per sample(uL)10End Prep Reaciton Volume6.011NEBNext Ultra II Ligation Master Mix3.012NEBNext Ligation Enhancer0.113Total Volume9.114&nbsp;&nbsp;15Adaptor Master Mix (1x)16Reagent(red tube)Volume per sample(uL)17Diluted Adaptor0.2518Note: Adaptor should be diluted based on approximate sample input and should not be added to adaptor ligation master mix to avoid adaptor-dimers.&nbsp;19&nbsp;&nbsp;20USER/PCR Master Mix (1x)21Reagent(USER- white tube; Q5- blue tube)Volume per sample(uL)22Adaptor Ligation Reaction Volume5.023Nuclease Free Water2.524NEB USER Enzyme0.925NEBNext Ultra II Q5 Master Mix7.526Total Volume15.9
?. [PREPARE MASTER MIX REAGENTS]
Spin the Echo source plate in the centrifuge for 5 minutes at 400g to rid source wells of any bubbles produced in pipette transfer of master mixes.
Time and rpm was per recommendation by Labcyte.Reagents must come to room temperature before dispensing on the Echo.
?. [DISPENSING END PREP REAGENTS]
For 384-well source plates, use the Echo's “Survey” setting to determine volume in each source well to ensure volume is 20
?. [DISPENSING END PREP REAGENTS]
Using the BP setting, dispense 1000nL of end prep master mix into each sample well.
Ensure there are no bubbles present in the source plates after spinning.
?. [DISPENSING END PREP REAGENTS]
Remove sample plate and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
?. [END PREP INCUBATION]
AB1End Prep Incubation2Heated Lid: &gt;75 °C3Temperature (°C)Time (mins)4203056530610Hold  A1End Prep Incubation 
2Heated Lid: &gt;75 °C
3Temperature (°C)
420
565
610
AB1End Prep Incubation2Heated Lid: &gt;75 °C3Temperature (°C)Time (mins)4203056530610Hold
A1End Prep Incubation 
2Heated Lid: &gt;75 °C
3Temperature (°C)
420
565
610
?. [DISPENSING ADAPTOR LIGATION REAGENTS ]
Remove sample plate from thermocycler, remove foil seal, and load into Echo.  Using the GP setting, transfer 3100nL of adaptor ligation master mix and 250nL of appropriately diluted adaptor to each sample well. Make sure to add these reagent separately; this will prevent excessive adaptor dimer.
?. [DISPENSING ADAPTOR LIGATION REAGENTS ]
Remove sample plate and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
?. [ADAPTOR LIGATION INCUBATION]
AB1Adaptor Ligation2Heated Lid: Off3Temperature (°C)Time (mins)42015510Hold  A1Adaptor Ligation
2Heated Lid: Off
3Temperature (°C)
420
510
AB1Adaptor Ligation2Heated Lid: Off3Temperature (°C)Time (mins)42015510Hold
A1Adaptor Ligation
2Heated Lid: Off
3Temperature (°C)
420
510
?. [USER/PCR SETUP - REAGENT PLATE]
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.
MUST be done before starting the bead clean-up.
?. [NUCLEIC ACID PURIFICATION: BEAD CLEAN]
Bead clean the end-prepped, adaptor-ligated cDNA on the Biomek using a sample to Ampure bead solution ratio of 0.8x.Use 5uM TRUESEQ BARCODES to elute sample from beads.  At least 10uL of barcode should be in each well of a 384-well PCR plate in order to elute correctly.Use USER/PCR master mix plate as final destination plate of bead clean-up.   AB1Post&nbsp;Adaptor Ligation&nbsp;Bead Clean (0.8x)2Bead Clean StepsVolumes per sample(uL)3Initial Sample Volume9.354Ampure Bead Volume7.285Elution Volume6.06Recover Volume5.0
AB1Post&nbsp;Adaptor Ligation&nbsp;Bead Clean (0.8x)2Bead Clean StepsVolumes per sample(uL)3Initial Sample Volume9.354Ampure Bead Volume7.285Elution Volume6.06Recover Volume5.0
USER/PCR master mix plate MUST be prepared before bead cleaning the sample. This plate is used to collect the final elution.
?. [USER/PCR PLATE PREPARATION]
Ensure FINAL PLATE of the bead clean plate contains:  A1sample cDNA2barcodes3USER/PCR master mixRemove from Bravo, and seal with a foil seal.  Gently vortex plate to mix, followed by quick spin.
A1sample cDNA2barcodes3USER/PCR master mix
?. [USER/PCR INCUBATION]
ABC1USER/PCR Incubation2Heated Lid: 105 °C3CyclesTemperature (°C)Time413715 m519830 s6&nbsp;199810 s76575 s81655 m9110Hold
ABC1USER/PCR Incubation2Heated Lid: 105 °C3CyclesTemperature (°C)Time413715 m519830 s6&nbsp;199810 s76575 s81655 m9110Hold
PCR cycle number is dependent on RNA input per sample in the first step of this protocol. For samples with undetectable RNA, we recommend a maximum of 19 cycles.
?. [NUCLEIC ACID PURIFICATION: BEAD CLEAN]
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.  AB1Post&nbsp;PCR&nbsp;Bead Clean (0.8x)2Bead Clean StepsVolumes per sample(uL)3Initial Sample Volume15.94Ampure Bead Volume12.725Elution Volume11.0 - 26.06Recover Volume10.0 - 25.0
AB1Post&nbsp;PCR&nbsp;Bead Clean (0.8x)2Bead Clean StepsVolumes per sample(uL)3Initial Sample Volume15.94Ampure Bead Volume12.725Elution Volume11.0 - 26.06Recover Volume10.0 - 25.0
Elutions volume may vary per application, but if contiuing to automated pooling step elute in 25uL and recover 24uL to account for Labcyte Echo dead volume.
?. [OPTIONAL NUCLEIC ACID PURIFICATION: BEAD CLEAN]
To remove additional primer- and adaptor-dimers, it may be necesssary to perform a second bead clean to remove the smaller fragments at 0.8x of Ampure beads to sample volume. This should be done after a QC to determine if there is significant dimers in libraries. Use Nuclease-free water to elute sample from beads.  AB12x Post PCR Bead Clean (0.8x)2Bead Clean StepsVolumes per sample(uL)3Initial Sample Volume104Ampure Bead Volume85Elution Volume11.0 - 26.06Recover Volume10.0 - 25.0
AB12x Post PCR Bead Clean (0.8x)2Bead Clean StepsVolumes per sample(uL)3Initial Sample Volume104Ampure Bead Volume85Elution Volume11.0 - 26.06Recover Volume10.0 - 25.0
Elution volume may vary depending on downstream applications, but if continuing to automated pooling step, elute in 25uL and recover 24uL to account for the Echo's dead volume.
?. [PREPARE MASTER MIX REAGENTS]
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.
?. [PREPARE MASTER MIX REAGENTS]
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.
For reagent master mixes that require more than 12 source wells of a 384-well Echo source plate (PP-200), it may be prudent to use a 6-well reservoir source plate (ER-0050) to minimize dead volume. For example, in this prep, samples require a large volume of second strand synthesis master mix.  Using a 6-well Echo reservoir plate for this step reduces dead volume and therefore requires less volume of reagents.
?. [FINISHED LIBRARIES]
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.
{"blocks":[{"key":"d66ip","text":"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 525 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.  For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"3gban","text":"","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"6ot5v","text":"After library preparation, the next step before putting samples on the sequencer is to pool samples to be able to multiplex sequencing. Final concentrations of each sample will vary from sample to sample depending on various things like RNA input, library prep, and PCR cycles among other factors. It is difficult to\u00a0manually quantify and pool uniformly 384 samples, so that is why we employ robots. To quickly and accurately pool hundreds of samples at a time, we employed a two-step method.\u00a0 Using the Labcyte Echo, equal volumes of each library are pooled together and sequenced on the Illumina iSeq or MiSeq sequencing platforms to determine the representation of each library in the total pool.\u00a0\u00a0Using this information, libraries were pooled using the Echo into a normalized pool for downstream sequencing on the Illumina NovaSeq.","type":"align-left","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [USER/PCR SETUP - REAGENT PLATE]
Load a new, sterile, empty PCR plate into the Echo destination port.  Using the GP setting, dispense 10,900nL of USER/PCR master mix into each sample well.
?. [NUCLEIC ACID PURIFICATION: BEAD CLEAN]