Methylation markers and targeted methylation probe panel

1. A method for processing cell-free DNA fragments, the method comprising (a) contacting converted cell-free DNA (cfDNA) molecules or amplification products thereof with a panel of different oligonucleotide probes configured to hybridize to converted cell-free DNA fragments derived from at least 500 target genomic regions; (b) enriching for probe-bound DNA to produced enriched DNA; and (c) sequencing the enriched DNA or amplification products thereof;

wherein the converted cfDNA molecules comprise deaminated nucleotides obtained by treating cfDNA molecules with a deaminating agent;

wherein each target genomic region of the at least 500 target genomic regions has an anomalous methylation pattern in cancerous samples; and

wherein each of the at least 500 target genomic regions comprises a sequence with complementarity to at least a pair of probes in the panel, wherein each probe of a pair has a first portion and a second portion, wherein the first portions of a probe pair are complementary to the same sequence of at least 25 nucleotides in length, and wherein the second portions of a probe pair are complementary to different sequences at different ends of the sequence complementary to the respective first portions.

2. The method of claim 1 , wherein each of the at least 500 target genomic regions is determined to be anomalously methylated in cancer training samples based on criteria comprising a number of cancer samples that comprise an anomalously methylated cfDNA fragment that overlaps the target genomic region, and wherein the target genomic region is determined to be anomalously methylated in cancer training samples relative to non-cancer training samples based on criteria comprising N cancer and N non-cancer , wherein:

N cancer , for each CpG, site is a number of cancer samples that include a cfDNA fragment covering the CpG site in the cfDNA fragment that (1) has at least 4 CpG sites, wherein at least 70% of the CpG sites are methylated or unmethylated and (2) has a p-value rarity in non-cancerous samples of below a threshold value; and

N non-cancer , for each CpG, site is a number of non-cancer samples that include a cfDNA fragment covering the CpG site in the cfDNA fragment that (1) has at least 4 CpG sites, wherein at least 70% of the sites are methylated or unmethylated and (2) has a p-value rarity in non-cancerous samples of below a threshold value.

3. The method of claim 2 , wherein each of the at least 500 target genomic regions is determined to be anomalously methylated based on criteria positively correlated with N cancer and negatively correlated with N non-cancer .

4. The method of claim 2 , wherein each of the at least 500 target genomic regions is determined to be anomalously methylated based on a score, wherein the score is calculated as (N cancer +1)/(N cancer +N non-cancer +2).

5. The method of claim 1 , wherein the at least 500 target genomic regions comprise at least 500 of genomic regions identified in List 1, List 2, List 3, List 4, List 5, List 6, or List 8.

6. The method of claim 1 , wherein for each probe of the pairs of probes, the first portion is at least 30 nucleotides in length, and the second portion is at least 15 nucleotides in length.

7. The method of claim 1 , wherein for each probe of the pairs of probes, the second portion is at least 50 nucleotides in length.

8. The method of claim 7 , wherein for each probe of the pairs of probes, the first portion and second portion together are at least 65 nucleotides in length.

9. The method of claim 7 , wherein each probe of the pairs of probes is less than 300 nucleotides in length.

10. The method of claim 1 , wherein the target genomic regions are human genomic regions, and further wherein at least one probe for each of the at least 500 target genomic regions (a) comprises a length of at least 45 bases, and (b) does not comprise a contiguous 45 bases with at least 90% sequence complementarity to 20 off-target regions in a GRCh37/hg19 genome.

11. The method of claim 1 , wherein each of the oligonucleotide probes in the panel is conjugated to an affinity moiety, wherein the affinity moiety is not a nucleic acid.

12. The method of claim 1 , wherein the mixture panel of different oligonucleotide probes is contacted to the converted cfDNA molecules.

13. The method of claim 1 , wherein the cfDNA molecules or amplification products thereof comprise adapter sequences at one or both ends.

14. The method of claim 13 , wherein the cfDNA molecules or amplification products thereof comprise a first adapter sequence at a first end and a second adapter sequence at a second end, wherein the first adapter sequence and the second adapter sequence are different.

15. The method of claim 13 , wherein (i) each of a plurality of the adapter sequences comprise a unique molecular identifier; and (ii) the method further comprises using the unique molecular identifiers to distinguish sequencing reads for different cfDNA molecules.

16. The method of claim 1 , further comprising, prior to said contacting, joining first adapter polynucleotides to a plurality of the cfDNA molecules.

17. The method of claim 16 , further comprising preparing the amplification products prior to said contacting by amplifying the cfDNA molecules joined to said first adapter polynucleotides to produce first amplification products comprising a first adapter sequence.

18. The method of claim 17 , wherein preparing the amplification products further comprises joining second adapter polynucleotides comprising a second adapter sequence to the first amplification products; and further wherein (i) the second adapter polynucleotides are joined to an end distal to the first adapter sequence of the first amplification products; and (ii) the first adapter sequence and second adapter sequence are different.

19. The method of claim 18 , wherein preparing the amplification products further comprises amplifying the first amplification products to produce second amplification products; and further wherein the second amplification products comprise (i) the first adapter sequence or a complement thereof, and (ii) the second adapter sequence or a complement thereof.