PicoPLEX™ DNA-seq Kit

Single Cell  Library Preparation for Illumina® NGS Platforms

PicoPLEX-DNA-seq Product Contents WEB-CROPPED
“Remarkably, the sequencing data from the PicoPLEX DNA-seq libraries of PGD embryos clearly showed two small, unbalanced segments consistent with the predicted patterns from high resolution FISH re-testing of a maternal blood sample that was initially scored as normal. This is a significant example of the sequencing data from embryos exposing a cryptic translocation missed by microarrays.”               

–Brian D. Mariani,
PhD, Chief Scientist, Scientific Director, Genetics & IVF Institute

Overview

PicoPLEX™, the technology used by IVF clinics worldwide for pre-implantation genetic screening and diagnosis in detecting chromosomal aneuploidies and copy number variations, is now available for use on your Illumina NGS platform!  PicoPLEX DNA-seq kit streamlines library preparation; the entire process is performed in a single tube or well – reducing error and contamination, speeding time to results, and reducing costs.  PicoPLEX DNA-seq kit contains 48 reactions and includes everything necessary to convert 48 individual cells or DNA (6 pg to 60 pg) to NGS libraries, including dual barcodes. Barcoding oligonucleotides are provided in a single-use microwell plate.

  • Reduce ambiguity–highly reproducible CNV and aneuploidy detection
  • Reduce workflow–from a single cell to a sequencing-ready library in three steps
  • Reduce cost–a single kit contains everything needed to prepare a sequencing-ready library
  • Reduce contamination and error–library prep in a single tube or well, no transfers necessary
  • Reduce time to results–Illumina NGS  libraries prepared  in  less than 3 hours

Workflow

PicoPLEX™-DNA-seq-Protocol-tubes-with-times-WEB

Protected by US Patent 8,206,913 and EU pending applications

 

Agencourt AMPure XP is a registered trademark of Beckman Coulter, Inc. MiSeq is a registered trademark and 24sure is a trademark of Illumina, Inc.

PicoPLEX DNA-seq Library Preparation Workflow for Illumina NGS Platforms

PicoPLEX DNA-seq Library Preparation Workflow for Illumina NGS Platforms
PicoPLEX DNA-seq Library Preparation Workflow for Illumina NGS Platforms

PicoPLEX DNA-seq Provides Aneuploidy Calls Concordant with Arrays

PicoPLEX DNA-seq Provides Aneuploidy Calls Concordant with Arrays
PicoPLEX DNA-seq Provides Aneuploidy Calls Concordant with Arrays

PicoPLEX DNA-seq was tested for equivalency to 24sure™ arrays (Illumina) by using the same single cell library for array and NGS testing. Sequencing results were concordant with array results and sequencing showed a terminal gain in 2p (shown in figure) and a terminal loss in 12q (data not shown), which were consistent with an undetected translocation in the paternal DNA confirmed by high resolution FISH analysis.” (Data provided by GIVF)

Highly Reproducible CNV Detection Over the Entire Genome

Highly Reproducible CNV Detection Over the Entire Genome
Highly Reproducible CNV Detection Over the Entire Genome

Amplified libraries from flow-sorted H929 cells were sequenced on an Illumina MiSeq® and downsampled to 250,000 total reads. Thirty-five base single-end reads were mapped to human over the entire genome.

Highly Reproducible CNV Detection in Chromosome 1

Highly Reproducible CNV Detection in Chromosome 1
Highly Reproducible CNV Detection in Chromosome 1

In chromosome 1, a 22 Mb loss and 30 Mb gain were consistently detected in each of the cells as indicated by the pink or blue bars.

Individual Library Quantification Eliminated

Individual Library Quantification Eliminated
Individual Library Quantification Eliminated

Flow-sorted unsynchronized H929 cells were amplified, pooled with constant volume, and loaded onto a MiSeq v3 flow cell. Quantification of the libraries was unnecessary before pooling due to the highly reproducible amount of product produced by PicoPLEX DNA-seq reactions. The columns which provided no reads were wells in which the cell was absent.

PicoPLEX Schematic

PicoPLEX Schematic
PicoPLEX Schematic

PicoPLEX™ DNA-seq uses the same technology as the WGA kit: cells are lysed, quasi-random primers pre-amplify the DNA selectively, and a final PCR amplification adds the Illumina barcodes.

Store at -20 °C.

Guaranteed for 12 months at -20°C in a constant temperature freezer.

Recommended DNA inputs:

  • Mammalian cells (1-10; e.g. blastomeres, polar bodies, trophoblastic cells, amniocytes, CTCs, cultured cells
  • DNA (6 pg- 60 pg)

Maximum Sample Volume, 5 µL

FAQs

1. What cell types have been successfully used with PicoPLEX DNA-seq?    
Single blastomeres, trophectoderm cells, and cultured mammalian cells have been used successfully.                                        

2. Which cell collection methods are compatible with PicoPLEX DNA-seq?
Flow sorting, dilution, and micromanipulation are compatible with PicoPLEX DNA-seq.

3. Should cells be washed before collection?
Yes, cells should be washed to minimize extracellular DNA or growth media contaminants. We recommend washing in PBS (Ca-free, Mg-free, and BSA-free) and limiting carryover of wash buffer to less than 2.5 µL.

4. Are there special requirements for flow sorting?
Yes, we strongly recommend not fixing the cells and using light scattering or phase contrast to sort or collect samples. Microscopic/visual confirmation of successfully sorted cells can be used to optimize sorting conditions. Surface antibody stained cells can be used if the cells are not fixed.

5. What is the sample input volume for PicoPLEX DNA-seq Kit?
This kit will accommodate 5 uL sample input volume per reaction with a maximum of 2.5 uL of cell & buffer carryover. If the cells are in 1x PBS, do not exceed 2.5 µL of PBS.

6. Do we have to separately purchase barcoding oligonucleotides? Can we use barcodes from other vendors?
Single use dual barcoding oligonucleotides in a 96-well format are included with the kit. Barcodes from other sources are not compatible with PicoPLEX DNA-seq.

7. Can we use the Dual Index Plate more than once?
We recommend designing your experiment for 48 samples; however, you can use the plate up to 4 times. Please refer to Appendix 2 in the Instruction Manual for details.

8. What is the size range of fragments expected with libraries prepared using PicoPLEX DNA-seq kit?
Libraries prepared using PicoPLEX DNA-seq kit results in a broad size range distribution of fragments, typically ranging from ~300 to 1000 bp total size (~200 to 900 bp insert size). Use 300 bp as the size for calculating the library concentration.

9. What concentration (pM) of the purified library should I load on MiSeq, v3?
With libraries made from a single cell using PicoPLEX DNA-seq, typically a good starting point for MiSeq, v3 is to load 16 pM using an estimated library size of 300 bp for calculation purposes. It is very important to add at least 5% PhiX DNA to the library prior to loading on the flow cell to achieve optimal diversity.

10. How many bases should I trim for the analysis of the sequencing reads?
The first 11 cycles of each read will contain quasi-random bases introduced during the PicoPLEX DNA-seq library preparation. For sequence alignment, either trim the initial 14 bases from each read or begin calibration and data collection at base position 15.

Catalogue no. R300381    PicoPLEX™ DNA-seq Kit contains everything needed to do 48 reactions, including  48 dual indexes in a single use 96-well plate.

Order directly from our STORE.

Performance

PicoPLEX DNA-seq Library Preparation Workflow for Illumina NGS Platforms

PicoPLEX DNA-seq Library Preparation Workflow for Illumina NGS Platforms
PicoPLEX DNA-seq Library Preparation Workflow for Illumina NGS Platforms

PicoPLEX DNA-seq Provides Aneuploidy Calls Concordant with Arrays

PicoPLEX DNA-seq Provides Aneuploidy Calls Concordant with Arrays
PicoPLEX DNA-seq Provides Aneuploidy Calls Concordant with Arrays

PicoPLEX DNA-seq was tested for equivalency to 24sure™ arrays (Illumina) by using the same single cell library for array and NGS testing. Sequencing results were concordant with array results and sequencing showed a terminal gain in 2p (shown in figure) and a terminal loss in 12q (data not shown), which were consistent with an undetected translocation in the paternal DNA confirmed by high resolution FISH analysis.” (Data provided by GIVF)

Highly Reproducible CNV Detection Over the Entire Genome

Highly Reproducible CNV Detection Over the Entire Genome
Highly Reproducible CNV Detection Over the Entire Genome

Amplified libraries from flow-sorted H929 cells were sequenced on an Illumina MiSeq® and downsampled to 250,000 total reads. Thirty-five base single-end reads were mapped to human over the entire genome.

Highly Reproducible CNV Detection in Chromosome 1

Highly Reproducible CNV Detection in Chromosome 1
Highly Reproducible CNV Detection in Chromosome 1

In chromosome 1, a 22 Mb loss and 30 Mb gain were consistently detected in each of the cells as indicated by the pink or blue bars.

Individual Library Quantification Eliminated

Individual Library Quantification Eliminated
Individual Library Quantification Eliminated

Flow-sorted unsynchronized H929 cells were amplified, pooled with constant volume, and loaded onto a MiSeq v3 flow cell. Quantification of the libraries was unnecessary before pooling due to the highly reproducible amount of product produced by PicoPLEX DNA-seq reactions. The columns which provided no reads were wells in which the cell was absent.

PicoPLEX Schematic

PicoPLEX Schematic
PicoPLEX Schematic

PicoPLEX™ DNA-seq uses the same technology as the WGA kit: cells are lysed, quasi-random primers pre-amplify the DNA selectively, and a final PCR amplification adds the Illumina barcodes.

Specifications

Store at -20 °C.

Guaranteed for 12 months at -20°C in a constant temperature freezer.

Recommended DNA inputs:

  • Mammalian cells (1-10; e.g. blastomeres, polar bodies, trophoblastic cells, amniocytes, CTCs, cultured cells
  • DNA (6 pg- 60 pg)

Maximum Sample Volume, 5 µL

Resources

FAQs

FAQs

1. What cell types have been successfully used with PicoPLEX DNA-seq?    
Single blastomeres, trophectoderm cells, and cultured mammalian cells have been used successfully.                                        

2. Which cell collection methods are compatible with PicoPLEX DNA-seq?
Flow sorting, dilution, and micromanipulation are compatible with PicoPLEX DNA-seq.

3. Should cells be washed before collection?
Yes, cells should be washed to minimize extracellular DNA or growth media contaminants. We recommend washing in PBS (Ca-free, Mg-free, and BSA-free) and limiting carryover of wash buffer to less than 2.5 µL.

4. Are there special requirements for flow sorting?
Yes, we strongly recommend not fixing the cells and using light scattering or phase contrast to sort or collect samples. Microscopic/visual confirmation of successfully sorted cells can be used to optimize sorting conditions. Surface antibody stained cells can be used if the cells are not fixed.

5. What is the sample input volume for PicoPLEX DNA-seq Kit?
This kit will accommodate 5 uL sample input volume per reaction with a maximum of 2.5 uL of cell & buffer carryover. If the cells are in 1x PBS, do not exceed 2.5 µL of PBS.

6. Do we have to separately purchase barcoding oligonucleotides? Can we use barcodes from other vendors?
Single use dual barcoding oligonucleotides in a 96-well format are included with the kit. Barcodes from other sources are not compatible with PicoPLEX DNA-seq.

7. Can we use the Dual Index Plate more than once?
We recommend designing your experiment for 48 samples; however, you can use the plate up to 4 times. Please refer to Appendix 2 in the Instruction Manual for details.

8. What is the size range of fragments expected with libraries prepared using PicoPLEX DNA-seq kit?
Libraries prepared using PicoPLEX DNA-seq kit results in a broad size range distribution of fragments, typically ranging from ~300 to 1000 bp total size (~200 to 900 bp insert size). Use 300 bp as the size for calculating the library concentration.

9. What concentration (pM) of the purified library should I load on MiSeq, v3?
With libraries made from a single cell using PicoPLEX DNA-seq, typically a good starting point for MiSeq, v3 is to load 16 pM using an estimated library size of 300 bp for calculation purposes. It is very important to add at least 5% PhiX DNA to the library prior to loading on the flow cell to achieve optimal diversity.

10. How many bases should I trim for the analysis of the sequencing reads?
The first 11 cycles of each read will contain quasi-random bases introduced during the PicoPLEX DNA-seq library preparation. For sequence alignment, either trim the initial 14 bases from each read or begin calibration and data collection at base position 15.

Ordering Information

Catalogue no. R300381    PicoPLEX™ DNA-seq Kit contains everything needed to do 48 reactions, including  48 dual indexes in a single use 96-well plate.

Order directly from our STORE.