The current protocol describes a method for DNA isolation from blood samples and intestinal biopsies, generation of TCRβ and IGH PCR libraries for next-generation sequencing, performance of a NGS run and basic data analysis.
This method enables exploration of specific T and B cells at the nucleotide or amino acid level and thus can identify dynamic changes in lymphocyte populations and diversity parameters in different disorders. The advantage of next-generation sequencing is the ability to identify unique T or B cell clones and track them in different anatomical sites or in different individuals. This has the potential for identification of novel biomarkers in diverse diseases.
Demonstrating that procedure will be Naomi Salamon, a research assistant from my laboratory. Begin by performing digestion and cell lysis of intestinal biopsies. Retrieve intestinal biopsies either freshly collected or stored at negative 20 or negative 80 degrees Celsius.
If using frozen biopsies, thaw them on ice. Add 600 microliters of nuclei lysis solution to a sterile 1.7-milliliter microcentrifuge tube, chilled on ice. Then place a biopsy into the tube, and incubate it at 65 degrees Celsius for 15 to 30 minutes.
Add 17.5 microliters of 20-milligrams-per-milliliter proteinase K, and incubate the tube overnight at 55 degrees Celsius, with gentle shaking. After the incubation, allow the sample to cool to room temperature for five minutes. For DNA isolation from blood, gently rock the tube until it is thoroughly mixed.
Then transfer blood to a sterile 15-milliliter centrifuge tube containing nine milliliters of cell lysis solution. Invert the tube several times during a 10-minute incubation period at room temperature. Centrifuge at 600g for 10 minutes at room temperature.
Then discard as much supernatant as possible without disturbing the visible white pellet. Vortex the tube vigorously for 15 seconds until completely resuspended. Add three milliliters of nuclei lysis solution, and pipette several times, causing the solution to become viscous.
Add protein precipitation buffer to the sample. Then vortex vigorously for 20 seconds, and incubate it on ice for five minutes. Centrifuge at 16, 000g for four minutes to form a tight pellet containing the precipitated proteins.
Carefully transfer supernatant without disturbing the pellet to a new 1.7-milliliter tube. Add one milliliter of 2-propanol to the tube containing the supernatant, and gently mix it by inverting. The DNA should become visible as a white floating substance.
Centrifuge at 16, 000 times g for one minute, and remove the supernatant completely. Add one milliliter of freshly prepared 70%ethanol, and invert the tube several times. Centrifuge it at 16, 000g for one minute at room temperature.
Then carefully discard the supernatant. Repeat the ethanol wash. Then leave the DNA pellet to air-dry for 10 to 15 minutes.
Add 50 microliters of ultra-pure water to the DNA, and perform DNA quantification as described in the text manuscript. Use 150 nanograms of DNA to prepare the library for sequencing. Place pipettes and tips in the hood with UV light for 15 minutes to break down residual DNA.
Meanwhile, allow the different index tubes and DNA polymerase to thaw on ice. In a biological hood, add 45 microliters from the different index tubes to the PCR tubes, taking care to avoid cross-contamination. Then add 0.2 microliters of the DNA polymerase and five microliters of the prepared DNA into the PCR tubes.
Run PCR using a preset program according to the manufacturer's instructions. Use magnetic beads for removal of excess primers, nucleotides, and enzymes. Warm the beads to room temperature for at least 30 minutes, and prepare enough fresh 80%ethanol for 400 microliters per sample.
Add the beads to each PCR tube, and mix by pipetting 10 times. Incubate the tubes for 10 minutes at room temperature. Place the mixed sample on the magnetic stand for five minutes.
Then aspirate the clear liquid, and discard. Aspirate the remaining liquid using a 10-microliter tip. Add 200 microliters of 80%ethanol to each sample, and incubate for 30 seconds.
Aspirate 195 microliters of ethanol, and discard. Then aspirate the remaining liquid using a 10-microliter tip. Repeat the ethanol wash.
Then open the caps of the tubes, and let them air-dry for five minutes. After five minutes, remove the samples from the magnetic stand, and add 25 microliters of elution buffer. Mix by pipetting until homogeneous, and incubate at room temperature for two minutes.
Place the tubes on the magnetic stand for five minutes. Then transfer 22 microliters to a new PCR tube, and measure the DNA concentration. Quantify the amplicon as described in the text manuscript.
After calculating the final DNA concentration and the size in base pairs of the product's peak, prepare a new dilution mix for each sample, and transfer two microliters of it to a new pool mix. Prepare a fresh solution of 0.2 normal sodium hydroxide, and add 10 microliters to the diluted library. Incubate for five minutes at room temperature to denature the double-stranded DNA.
Then add 980 microliters of pre-chilled buffer from the kit to the tube containing DNA, and vortex briefly. Load 600 microliters of the final library onto the designated cartridge, and place the samples in the machine. To perform sequencing analysis, download sequencing metrics from the sequencer, and verify that the data is within the appropriate ranges.
Repeat the run for samples that do not meet the criteria. This protocol was used to perform a basic analysis of representative autologous blood and rectal samples of a patient with IL10 receptor deficiency and a history of severe infantile-onset IBD. Samples were assayed for both TCR-beta and IGH repertoire.
All clones can be identified by their unique sequence at the nucleotide or amino acid level. These sequences can be compared between different patients in search for shared clones or in the same patient between different anatomical sites. The five most frequent clones for each sample are shown here.
TreeMap images were generated for a broad overview of the repertoire. Each colored square represents a different clone, and the size correlates with its frequency. These TreeMap images demonstrate clonal expansion in the intestinal IGH repertoire.
In contrast, in the TCR-beta repertoire, marked clonal expansion is observed in the blood in comparison to the autologous intestine. At the gene level, NGS provides information regarding V, D, and J usage at the level of either gene, family, or allele. Moreover, specific VDJ combinations can be inferred from the data for TCR-beta and IGH repertoires, revealing differential gene usage patterns in various conditions.
This protocol can be applied on both DNA and RNA and can be used to generate TCR-alpha, TCR-gamma, and IGL repertoires. It is also compatible with other organs after slight digestion modifications.
