Utilizing the Labcyte Echo® Liquid Handler to Validate the Generation of Recombinant Cell Lines by Next-Gen SequencingJohn Lesnick, Jefferson Lai, Noël RuppertLabcyte Inc. Sunnyvale, CA, USA

© 2017 LABCYTE INC. All rights reserved. Labcyte® and Echo® are registered trademarks of Labcyte Inc., (“Labcyte”) in the United States and/or other countries.

Presented at SLAS2017

POS-JLUE-1.0JAN 2017

#1303

IntroductionThe study of protein function relies upon recombinant gene assembly for protein expression and

functional assays. This strategy is particularly important when available information regarding function

is limited. The vast amount of data generated from genome sequencing projects has paved the way

for diverse functional genomic studies that involve cloning, modification and subsequent expression

of target genes. This requires flexible and efficient procedures for generating vectors containing gene

fusions, gene variants, genetic coding for protein tags and cellular targeting. Furthermore, efficient

high-throughput cloning and gene assembly procedures are essential for interrogating signaling

pathways with multiple gene constructs. The resulting gene constructs can be analyzed in stable

transgenic or in transiently expressing biological systems and confirmed by NGS.

Utilizing the Labcyte Echo 525 liquid handler we demonstrate an efficient and robust gene assembly

method to generate a fusion protein for functional cellular assays. A PTEN-GFP construct was generated

using the Gateway chemistry. PTEN null PC-3 cells were transfected with this construct to generate a

fluorescently-labelled PTEN cell line to interrogate the PI3K pathway. The DNA from these cells was

purified and the construct integration into the genome was confirmed by whole genome sequencing.

The assembled fusion protein now enables functional cellular assays including, but not limited to,

viability and localization studies. The Echo 525 liquid handler enables flexibility and scalability for gene

assembly while streamlining and automating the process. The library preparation was miniaturized and

streamlined for automation by using the Echo System’s acoustic, non-contact dispensing eliminates

tips and cross-contamination. The low 25 nL resolution enables reduction in reaction volume which

results in reduced reagent consumption and reduced costs for the user.

Materials and MethodsGene Assembly and TransfectionPC-3 Cell Line (ATCC # CRL-1435)

Growth Media (RPMI 1640 supplemented with 10%FBS and 100 U/ml Pen/Strep)

Gateway PLUS shuttle clone for PTEN(NM_000314.4) (Purified plasmid)

(Genecopoeia # GC-I0450-CF) Entry Plasmid

Gateway® pcDNA™-DEST47 Vector (Fisher #12281010) Destination Plasmid

Gateway® LR Clonase® II Enzyme mix (Fisher #11791020)

Proteinase K, recombinant, PCR grade (Fisher #EO0491)

Geneticin® Selective Antibiotic (G418 Sulfate) (Fisher #10131035)

Turbofectin 8.0 (Origene # TF81001)

Materials and MethodsWhole Genome Sequencing by NexteraFor detailed materials and methods for the Echo liquid handler miniaturization and streamlining of the

Nextera library preparation for whole genome sequencing, see Poster #1302 SLAS 2017, Labcyte Booth #523 or www.labcyte.com.

• Incubate 25oC 15hrs

• Add 200 µL growth media to reaction well

• Add 30 µL Turbofectin to reaction well and mix gently

• Incubate 25oC 15minutes

• Transfer reaction well to cells at 50% confluence

• Incubate humidified 5% CO2 incubator 48hrs

• Replace media with growth media plus G418 selection

• Image cells for GFP expression (EVOS cell imaging system)

To streamline the process and enable an automatable workflow, the transformation, negative selection

and isolation step(s) have been removed.

Labcyte Echo® Acoustic Liquid Handling

Summary• Gene assembly experiments miniaturized using the Echo liquid handler yield comparable data to

the manufacturer’s suggested reaction volumes and therefore, provide a significant cost savings to the end user. High-throughput cloning efforts could greatly benefit from this scale-down.

- Smart DNA Fabrication Using Sound Waves: Applying Acoustic Dispensing Technologies to Synthetic Biology Kanigowska et al. JALA Publication 2016, DOI: 10.1177/2211068215593754

• Transfection optimization with the Echo liquid handler can reduce development time and cost toward optimal DNA concentration and transfection reagent.

- Optimising cell line generation with acoustic dispensing https://www.researchgate.net/publication/308078112

- Acoustic Liquid Handling for Rapid siRNA Transfection Optimization DOI:10.1177/1087057115583808, jbx.sagepub.com

• The Echo Liquid Handler is shown to miniaturize the tagmentation reaction and PCR amplification for NGS Nextera library preparation resulting in significant cost and time savings.

• An extension of this study will include CRISPR-Cas 9 generated cell lines and screens for target identification and validation.

ResultsThe pooled libraries were prepared according to the standard procedure for sequencing with the NextSeq®

High-Output, paired end read 2 x 150 bp kit. HISAT2 (v2.04), a graph-based alignment of next generation

sequencing reads, was used to align sequence results to the human reference genome (NCBI hg38)

and generate quality metrics. The sequencing and informatics were run at SeqMatic (Fremont, CA).

FIGURE 1: Echo Liquid Handler Workflow

6 μL Entry Plasmid

2 μL Clonase II

2 μL Destination Plasmid

FIGURE 2: The Labcyte Echo 525 Liquid Handler

The Labcyte Echo 500 series liquid handlers

revolutionize liquid transfer by using acoustic energy

to eject fluids. Transfer with Echo Liquid Handlers

is completely touchless—no tips or nozzles, and no

material contacts the sample as it moves from source

to destination. This protects the integrity of samples

and precious reagents while providing additional cost

savings and eliminating waste, carry-over effects and

cross contamination. The Echo 525 Liquid Handler can

transfer in 25 nL increments to allow miniaturization

with accuracy and precision.

FIGURE 3: Transfected cells were grown under standard conditions for two weeks and imaged with the Evos cell imaging system. GFP expressing PC-3 cells under 1000ug/mL G418 selection demonstrated positive and stable transfection of recombinant vector. PTEN_GFP fusion protein is generally localized to the plasma membrane.

Results

TABLE 1: DNA input and miniaturized reaction volumes modified from Nextera using the Echo liquid handler. Sample input has been reduced more than 10-fold to 2.5 ng. The tagmentation reaction has been reduced to 2.5 µL and PCR amplification to 5.5 µL. The Zymo cleanup has been replaced with a 0.1% SDS treatment to remove tagmentase.

FIGURE 4: Electropherogram of library preparation measured by TapeStation. Library was quantified by Qubit prior to pooling and sequencing.

FIGURE 5: PTEN Gene Coverage Overview via Integrated Genomics Viewer (IGV) There are a total of 115 mapped reads resulting in a coverage depth of 9.6X. 100% of the gene is covered at 1X while 95% is covered at 5X.

Sample Sample Input (ng)

Tagmentation Volume (μL)

Tagmentation Removal

Elution Volume (μL)

PCR Volume (μL)

Final Elution (μL)

SDS 2.5 2.5 2.5 SDS 3.25 5.5 22

TABLE 2: Sequencing metrics for the Echo 525 liquid handler prepared library from recombinant PC3_GFP cells.

Percent Aligned

Percent Q30 Mismatch Rate Fragment

LengthPercent

Duplicate %GC Coverage

92% 95% 0.05% 236 12% 43% 10

TABLE 3: Coverage of PTEN ORF stably expressed in PTEN null PC-3 cells. Sequence reads were compared to the PTEN ORF to detect presence of inserted gene. The assembled construct does not contain a stop sequence in exon 9 and is fused to GFP resulting in failed alignment. Functional cellular assays will be run to confirm activity of PTEN and not a truncation.

exon1 exon2 exon3 exon4 exon5 exon6 exon7 exon8 exon9

10.11 12.34 10.02 6.89 9.1 13.4 8.22 9.89 0.21