With this method, we can accurately determine the number of mutations in a single hematopoietic stem cell. These mutations can be used to decipher mutational processes and dissect hematopoietic lineages. Our method avoids the use of whole-genome amplification techniques by cloning the hematopoietic stem cells in vitro, which results in lower error rates and benefits downstream analysis.
The mutational patterns present in the genomes of hematopoietic stem cells can reveal the mutagenic and the DNA repair processes to which they were exposed. Begin this procedure with preparation of sample material and staining as described in the text protocol, then prepare 25 milliliters of hematopoietic stem and progenitor cell, or HSPC culture medium. Fill a cell culture 384 well-plate with 75 microliters of HSPC culture medium in each well.
To prevent evaporation of the medium in the outer wells, fill the outer wells with 75 microliters of sterile water or PBS, and do not use these wells for cell sorting. Correct sorting of viable signal HSPCs is the most critical step in this protocol. Set gates for the HSPC sorting based on an unstained control in 10, 000 cells from the stained sample.
Gate single cells by drawing a gate around the linear forward scatter height versus forward scatter area fraction. Use the unstained control fraction to draw a gate for the lineage fraction. Draw gates for CD34 positive cells and further characterize this subset by setting a specific gate for CD38 negative, CD45RA negative cells.
Load the 384-well plate on the fax machine and sort single cells. If applicable to the fax machine, toggle on the option to keep index sorting data to enable retracing of the sorted cells. To culture singly sorted HSCs, wrap the 384-well culture plate with lid in transparent polyethylene wrap.
Transfer the 384-well plate to a humidified 37 degrees Celsius incubator with 5%carbon dioxide and keep it in the incubator for three to four weeks until visible clones appear. After four weeks of culturing, determine which wells have a confluency of 30%or higher. For each cone, prefill 1.5 milliliter microtubes with one milliliter of 1%BSA and PBS and label the tube according to the corresponding well.
Pre-wet a pipette tip with 1%BSA and PBS to minimize the number of cells sticking to the pipette tip. Vigorously pipette the medium up and down at least five times while scraping the bottom of the well with the 200 microliter pipette set at 75 microliters. Collect the cell suspension in the labeled microtube that corresponds to the well.
Repeat pipeting in the well with up to 75 microliters of fresh 1%BSA and PBS to ensure maximum uptake of cells. Clonally cultured cells can stick to the bottom of the well. Inspect the wells using a standard, inverted light microscope to ensure whether all cells have been collected.
If all wells with greater than 30%confluency have been harvested, place the 384-well plate back in the incubator. Clonal cultures can proliferate for up to five weeks. Spin down the cell suspension for five minutes at 350 times g.
A small pellet should be visible. Carefully remove all but about five microliters of the supernatant. Cell pellets can be frozen at minus 20 degrees Celsius and stored for multiple months before DNA isolation.
Mutations present in the first branches of the lineagery will also be sub clonally present in the bulk sample. Later branching lineages are defined by mutations shared between HSPCs only. To identify mutations that are present in a subset of the clones and subclonaly present in the bulk, first filter for somatic mutations shared between clones.
In a Unix based terminal, edit the filtersomatic. ne file to set the pats and adjust the other parameters. Run the filtersomatic.
pi script. Filter for mutations that are subclonaly present in the bulk using the determine lowVAF bulk. r script in a Unix based terminal.
This will generate separate vcf files for shared and unique single nucleotide variants. Determine all mutations shared between clones that are not present in the bulk sample, by overlapping all mutation positions. Exclude false positives by manual inspection using Integrative Genomic Viewer, abbreviated IGV.
Mutations are considered false when not present when the mutation is present in the germline in all clones or when present in poorly mapped regions. True mutations can be shared amongst two clones or be subclonaly present in the bulk. Resequence all shared loci independently using targeted or sanger sequencing.
Use the shared mutations to build a binary table of mutations versus sequenced clones, with zero indicating that the mutation is not present and one indicating presence of the mutation. Output the mutation binary table as a heat map together with a dendrogram indicating lineage relationships between cells using R based functions. The heat map indicates mutation status for each cell.
Shown as an example output of the copy number analysis generated by control three c to check for copy number alterations. The variant allele fraction plot created by single nucleotide variant filtering, is a histogram of variant allele frequencies in the sample. A peak in the density plot at 0.5 indicates the sample is clonal.
Depicted here is a typical mutational patterns analysis producing a 96 trinucleotide plot. In addition to quantification of different mutation types, signature extraction can be also performed with this tool. Mutations shared amongst clones or present in a clone in the germline control are validated using IGV.
Mutations are considered true when present in the sample and not at high variant allele frequency levels in the germline. Mutations are considered false when not present in IGV, which can happen in poorly mapped regions. In other cases, events detected by single nucleotide variant filtering are missed germline variants.
Independent resequencing of mutations by targeted resequencing is highly recommended for these mutations in selected clones. After detection of shared somatic mutations between clones, a binary matrix is generated, a heat map is constructed containing cells with and without the shared mutations A through M.The developmental lineage tree is indicated. As a next step for the meta presented here, mutational processes active in hemopoietic stem cells can be determined using mutational signature analysis.
