Method for measuring mutational load
Provided herein is method for, among other things, estimating the number of sequence variations in a sample of DNA. In some embodiments, the method can be used to estimate the mutational load of a sample. In some embodiments, the method makes use of a set of primers that have 3′ ends that specifically hybridizes to a sequence that is repeated multiple times in the genome. Thermocycling a reaction mix containing the primers may produce a reaction product comprising at least 50 amplicons having a total length of at least 100 kb. This product can be sequenced to provide an estimate of the number of sequence variations in the sample, and thus the mutational load of the sample.
1. A method for sequencing at least 100 kb of a genome comprising:
(a) combining cell-free DNA (cfDNA) from a patient with a thermostable polymerase, dNTPs and a primer set consisting of 10 to 100 different primers to produce a reaction mix, wherein the 3′ end of each primer specifically hybridizes to a sequence that is repeated at least 100 times in the genome of the patient;
(b) thermocycling the reaction mix to produce a reaction product comprising at least 500 amplicons having a total length of at least 100 kb; and
(c) sequencing the amplicons of step (b) or amplification products thereof to produce sequence reads.
2. The method of claim 1 , wherein the 3′ terminal 12 nucleotides of each primer is repeated at least 500 times in the genome of the patient.
3. The method of claim 1 , wherein the 3′ terminal 12 nucleotides of each primer is repeated at least 1,000 times in the genome of the patient.
4. The method of claim 1 , wherein the reaction product of step (b) comprises at least 1,000 amplicons covering at least 250 kb.
5. The method of claim 1 , wherein the reaction product of step (b) comprises at least 10,000 amplicons having a total length of at least 2.5 Mb.
6. The method of claim 1 , wherein amplicons of (b) are less than 500 bases in length.
7. The method of claim 1 , wherein the patient has non-small cell lung cancer (NSCLC), small cell lung cancer, bladder cancer, head and neck cancer, melanoma, renal cell carcinoma or a lymphoma.
8. The method of claim 1 , further comprising:
(d) analysing the sequence reads of (c) to estimate the number of sequence variations in the regions amplified in step (b).