Detection of Copy Number Alterations Using Single Cell Sequencing

The overall goal of this procedure is to identify megabase scale DNA copy number alterations in single cells. In order to categorize genomic heterogeneity in cell populations, it is necessary to identify genomic changes at single cell resolution. Traditionally, this was achieved using cytologic approaches, however, these methods were limited in their sensitivity and specificity.

Single-cell sequencing can detect a variety of genomic changes, with enhanced sensitivity and specificity over prior approaches, but only when carefully executed. Here, we describe how to use single cell sequencing to reliably detect megabase-scale copy number alterations in single cells. To assemble the micro-aspirator, begin by removing the clear plastic end from the aspirator tube assembly and insert the narrow end into one end of a one foot long PVC tubing with a 3/16th inch inner diameter.

Insert the outlet of a 0.2 micrometer syringe filter into the other end of the one foot long PVC tubing with a 3/16th inch inner diameter. Then insert the inlet of the 0.2 micrometer syringe filter into one end of a six inch long PVC tubing with a 5/16th inch inner diameter. Break a five milliliter plastic serological pipette at the one milliliter graduation and insert the broken end into the open end of the PVC tubing with the 5/16th inch inner diameter.

Next, insert the outlet of the five milliliter plastic serological pipette into the flexible end of an aspirator tube assembly, then use a sterile plastic tube to cover the red mouthpiece of the tube assembly for storage. Draw out a glass capillary tube to an inner diameter of 10-30 micrometers by positioning the middle of the tube near a flame and applying tension on either end of the tube. Break the drawn tube into two halves to create two potential aspirator needles.

Repeat this step with several tubes to ensure that enough needles are prepared with an appropriate inner diameter for picking single cells. To prepare adherence cells, such as human fibroblast cell lines, harvest cells by trypsinization, and transfer the cells to a conical tube containing the appropriate medium. Set up a hood for whole genome amplification by using 10%bleach to spray down the surface of the hood, pipette tip boxes, and pipette tips.

Then use a paper towel to wipe them all down before repeating the wash with 70%ethanol. Add eight microliters of water from the Whole Genome Amplification, or WGA, kit to individual wells to a 96-well PCR plate. Then cover the 96-well PCR plate with a lid from a 96-well tissue culture plate, and place the plate on ice.

Next, add 1, 000 cells to 10 milliliters of medium or PBS in a 15 centimeter Petri dish and place the dish on ice to prevent the cells from adhering to the dish. Then, while still on ice, transfer the cells in the Petri dish and the 96-well PCR plate to a light microscope with a 10X objective. Increase the opening of the aspirator needle by gently tapping the drawn-out end of the needle on a hard surface so that the tip breaks off.

Then insert the wide end of the needle into the clear end of the micro-aspirator. Next, place the Petri dish containing cells on the microscope stage. Insert the red mouthpiece of the aspirator in the mouth.

Then, while using one had to move the aspirator needle, and the other hand to move the Petri dish, identify single cells to be sequenced. Using mouth suction, draw a single cell into the aspirator needle along with approximately one to two microliters of medium or PBS. Transfer the cell into the eight microliters of water in a single well of the 96-well PCR plate.

The quality and utility of single cell sequencing data is dependent on the state of each cell and its genome therefore, take care during cell preparation and micro-aspiration to isolate viable and non-apoptotic cells. After aspirating each cell and transferring it to a well of the PCR plate, mark the well that has received a cell. Repeat this for the desired number of cells.

In the meantime, thaw 10X Single Cell Lysis Fragmentation Buffer from the Whole Genome Amplification kit. To prevent contamination during whole genome amplification, add all reagents inside a tissue culture hood. Use pipette tips with filters, and change the pipette tip in-between wells.

For each set of up to 32 cells, combine 32 microliters of 10X Single Cell Lysis and Fragmentation Buffer and two microliters of Proteinase K solution in a microcentrifuge tube, then vortex the tube to mix the contents. Add one microliter of the solution to each well and pipette up and down to mix. Cover the plate and use plastic film to seal all the wells.

Then briefly centrifuge the plate in a mini plate-spinner. Next, run the plate through the thermocycler using the following program. Then cool the plate on ice, and briefly centrifuge it.

To prepare a working library preparation buffer solution from the Whole Genome Amplification kit, for each cell, combine two microliters of 1X Single Cell Library Preparation Buffer, and one microliter of Library Stabilization Solution. Adequate cell lysis and complete genome fragmentation is critical in order to minimize bias during whole genome amplification and ensure preparation of high-quality sequencing libraries. As such, take care to perform these steps exactly as written.

Remove the plastic film from the dish and add three microliters of the solution to each well. Pipette the contents of the well up and down to mix, then replace the plastic film. After briefly spinning the plate, incubate the cells at 95 degrees Celsius for two minutes, then cool the plate on ice, and briefly centrifuge the plate again.

Now, remove the plastic film, add one microliter of Library Preparation Enzyme to each well, and pipette the contents of the well up and down to mix, then replace the plastic film. After briefly spinning the plate, place it in the thermocycler and run the following program. After cooling the plate and giving a brief spin, prepare a working amplification mix from the Whole Genome Amplification kit by combining 48.5 microliters of water, 7.5 microliters of 10X amplification master mix, and 5 microliters of WGA DNA polymerase, for each cell.

Keep the working mix on ice. Remove the plastic film from the plate. Add 61 microliters of the working amplification mix to each well, and pipette the contents of each well up and down to mix, then replace the plastic film.

Briefly centrifuge the plate and thermocycle as follows. Sequence the samples and carry out data analysis according to the text protocol. As demonstrated in this figure, if whole genome amplification is successful, the sample will appear as a smear on an agarose gel.

A faint or absent smear indicates a failed amplification reaction and the sample should not be sequenced. As shown here, analysis of the fragment sizes was carried out using capillary electrophoresis on a fragment analyzer. Successfully prepared libraries will have a rather even distribution of fragment sizes from 150 to 900 base pairs.

Failed library preparation will result in a skewed fragment size distribution and such libraries should not be sequenced. Using the hidden Markov model and circular binary segmentation, the genome of each cell was parsed into segments of estimated copy number. This table shows the overlapped results from a single cell and reveals a gain on chromosome 10 from 67 to 130 megabase, and a gain on chromosome 19 from 0 to 20 megabase.

The isolation and whole genome amplification of single cells can and should be performed on a single day. The preparation of sequencing libraries, whole genome sequencing, and data analysis, can then be performed on a flexible schedule. When cell isolation and whole genome amplification are performed properly, and data analysis is performed rigorously as described, copy number alterations exceeding five megabases can be detected with high sensitivity and specificity.

While our protocol specifically describes the use of single cell sequencing for the detection of copy number alterations, aspects of this protocol, such as single cell isolation and library preparation, can be applied to detect other genomic changes.