Skip to main content

Our sequencing explained

20150317_anthony_nolan_0537

We use Next Generation Sequencing to tissue type the HLA genes of our donor and patients. It allows us to determine the entire DNA sequence for each gene with unprecedented accuracy so we can be confident of finding the best possible donor for every patient.

How do you carry out HLA typing? 

HLA is a group of highly polymorphic genes in a region called the major histocompatibility complex, found on chromosome six. 

HLA typing involves identifying these gene sequences.  We analyse the DNA sequence of each gene, which allows us to define specific alleles and determine a person’s HLA type. 

DNA can come from a blood, buccal swab or saliva sample. We use a state-of-the-art method known as Next Generation Sequencing (NGS) for our HLA typing. 

There are several purification stages and quality checks along the way, and the whole process takes 3-4 days from receiving the DNA samples to the results. 

The process starts with a ‘block’ of DNA samples – patient or donor samples and control samples – extracted and prepared by the Technical Services Team so that the samples are normalised to the same concentration. 

Next generation sequencing (NGS) process: 

Using NGS we can determine the DNA sequence of the HLA genes from a simple blood, saliva or swab sample.

The process is characterised by six main steps:

Amplification 

The Typing and Development Team (TDT) start by amplifying the 11 HLA genes of interest. These are HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4 and -DRB5. The samples are amplified using a process called PCR, (polymerase chain reaction), creating millions of copies of the desired HLA genes for each sample.

Library preparation  

During PCR, the genes of interest (loci) within each sample are amplified separately. The first step in the library preparation is the pooling stage. This is when all the loci for each sample are combined. 

Fragmentation, end repair and adapter ligation 

At this step, restriction enzymes are applied to fragment the DNA. This happens because, for sequencing to work, the DNA fragments need to be a specific range of sizes. 

The DNA is randomly fragmented to ensure complete coverage of the amplified gene is achieved. It involves two enzymes, the first one generates nicks in the double stranded DNA and the second recognises the nicked sites and cuts the opposite strand of DNA across from the nick, producing a DNA break. 

In preparation for adapter ligation, the DNA fragments undergo end repair. Then adaption ligation takes place and adapters bind to the dA-tail of the DNA fragments, giving the primers in the sequencing process a site to attach onto. 

Indexing 

Indexing is completed after a series of cleaning and size selection steps. During indexing, each sample is given two indices i5 and i7 (similar to a barcode), which attaches onto the ends of the adapter region of the DNA fragment. The combination of these indices gives each sample a unique code and allows them to be identified, when they are all pooled together for the sequencing process. The indices also have a region called p5 and p7 sites, which are complimentary to the oligonucleotide binding sites on the flow cell, meaning that the DNA fragments can attach onto the flow cell for the sequencing process.

Loading the MiSeq 

Once the samples have undergone further cleaning and dilution steps. They are ready to be loaded onto the Illumina cartridge. This cartridge holds most of the reagents which are necessary for cluster amplification and sequencing by synthesis. The cartridge is then loaded into the MiSeq sequencer, alongside the flow cell.  

Sequencing  

The flow cell is critical in sequencing. It is a glass slide with lanes, consisting of a ‘lawn’ of two types of oligonucleotide binding sites, which allow for the DNA strands to attach. Once bound, an enzyme (DNA polymerase) will bind to the template DNA and create a complimentary copy of the DNA fragment by adding deoxynucleotide triphosphates (dNTPs), the building blocks of DNA, to it. The double stranded DNA is then denatured, and the original copy is washed away. At this point, single DNA strands fold over and each end is bound to an oligonucleotide to create a bridge. The DNA polymerase will bind to the template DNA again and create another complimentary strand. This is called bridge amplification. The process is then repeated multiple times creating clonal clusters. All reverse single DNA strands are cleaved, preparing the template DNA strands for sequencing by synthesis. 

At this point, fluorescent dNTPs are attached to the template DNA strand. As each individual DNA base (A, C, T or G – the four different chemicals that make up all our DNA) is added to the new DNA strand, a flash of light specific for each base is emitted. These flashes are picked up by two MiSeq cameras. This process is repeated in a total of 150 cycles (one dNTP per cycle) to complete the sequence of the first read (150bp). The dNTPs are then washed away, the template DNA undergoes bridge amplification again, this time the forward DNA strands are cleaved and sequencing by synthesis is repeated on the reverse strand (another 150 cycles). 

Currently we routinely sequence 24 samples at once, however the MiSeq instrument can run up to 96 samples, each containing 11 HLA genes – that’s hundreds of copies of 1,056 genes per run. 

All that data is processed and sorted by a program called NGSengine. A scientist can then analyse and review the data, so as an end product we have 11 loci of high to allelic resolution HLA typing for our samples. 

Category
Research