Single-molecule sequencing of plasma DNA

1. A method comprising:

receiving a biological sample obtained from a single subject, the biological sample including a bodily fluid comprising cell-free DNA fragments;

extracting a plurality of the cell-free DNA fragments from the biological sample;

concatemerizing, in a container, a first set of the plurality of the cell-free DNA fragments to obtain a first concatemer consisting of the first set of the plurality of the cell-free DNA fragments and optionally spacer DNA fragments, wherein the cell-free DNA fragments of the first set have different sequences and are from different locations in the genome of the subject;

concatemerizing a second set of the plurality of the cell-free DNA fragments to obtain a second concatemer consisting of the second set of the plurality of the cell-free DNA fragments and optionally spacer DNA fragments, wherein the second concatemer comprises a different combination or a different permutation of the cell-free DNA fragments than the first concatemer, wherein concatemerizing the second set of the plurality of the cell-free DNA fragments occurs in parallel in the container with the concatemerizing of the first set of the plurality of the cell-free DNA fragments;

performing single-molecule sequencing of the first concatemer to obtain a first sequence, the first sequence including the different sequences of the first set of the plurality of the cell-free DNA fragments as subsequences within the first sequence; and

performing single-molecule sequencing of the second concatemer to obtain a second sequence, the second sequence including the different sequences of the second set of the plurality of the cell-free DNA fragments as subsequences within the second sequence.

2. The method of claim 1 , wherein performing single-molecule sequencing of the first concatemer comprises providing the first concatemer to a sequencing device, wherein the sequencing device includes a first nanopore, and the method further comprises:

passing the first concatemer through the first nanopore; and

detecting first electrical signals as the first concatemer passes through the first nanopore, the first electrical signals corresponding to the first sequence of the first concatemer.

3. The method of claim 2 , further comprising performing, by a computer system:

analyzing the first electrical signals to determine the first sequence; and

aligning subsequences of the first sequence to a reference genome to identify fragment sequences corresponding to each of the first set of the plurality of the cell-free DNA fragments.

4. The method of claim 2 , wherein passing the first concatemer through the first nanopore includes:

passing a first strand of the first concatemer through the first nanopore; and

subsequently, passing a second strand of the first concatemer through the first nanopore.

5. The method of claim 4 , further comprising

analyzing, by a computer system, the first electrical signals for the first strand and the second strand to determine the first sequence.

6. The method of claim 2 , wherein the first nanopore is one of a plurality of nanopores on a substrate.

7. The method of claim 6 , further comprising:

passing the second concatemer through a second nanopore of the plurality of nanopores; and

detecting a plurality of second electrical signals as the second concatemer passes through the second nanopore, the second electrical signals corresponding to the second sequence of the second concatemer.

8. The method of claim 1 , further comprising:

hybridizing a fluorescent-labeled nucleotide to the first concatemer, and

detecting a fluorescent signal, the fluorescent signal corresponding to a nucleotide.

9. The method of claim 1 , further comprising performing, by a computer system:

aligning subsequences of the first sequence to a reference genome to identify fragment sequences corresponding to each of the first set of the plurality of the cell-free DNA fragments.

10. The method of claim 9 , further comprising:

determining a size of each of the first set of the plurality of the cell-free DNA fragments based on the aligning of the subsequences of the first sequence, wherein the first sequence does not include spacer DNA fragments.

11. The method of claim 9 , wherein:

concatemerizing the first set of the plurality of the cell-free DNA fragments comprises concatemerizing the first set of the plurality of the cell-free DNA fragments and the spacer DNA fragments, wherein the spacer DNA fragments are a set of DNA fragments having known sequences and wherein the set of DNA fragments having known sequences is interspersed among the first set of the plurality of the cell-free DNA fragments, and

aligning subsequences of the first sequence comprises aligning subsequences to the known sequences to identify locations of the set of DNA fragments having known sequences in the first concatemer.

12. The method of claim 11 , wherein the concatemerizing to form the first concatemer places one DNA fragment having a known sequence between two DNA fragments of the first set of the plurality of the cell-free DNA fragments.

13. The method of claim 12 , further comprising performing, by the computer system:

identifying, by the computer system, a starting base of a first subsequence corresponding to a first DNA fragment of the first set of the plurality of the cell-free DNA fragments based on identifying one of the DNA fragments having a known sequence being before the first subsequence; and

identifying, by the computer system, an ending base of the first subsequence corresponding to the first DNA fragment of the first set of the plurality of the cell-free DNA fragments based on identifying one of the DNA fragments having a known sequence being after the first subsequence.

14. The method of claim 11 , wherein each known sequence of the set of DNA fragments having known sequences has a length of no more than seven nucleotides.

15. The method of claim 11 , wherein each of the second set of the plurality of the cell-free DNA fragments has the same known sequence.

16. The method of claim 9 , wherein the aligning of the subsequences includes:

aligning sliding windows of the first sequence to the reference genome, each sliding window corresponding to a subsequence that is being aligned to the reference genome; and

identifying when two sliding windows align to different regions of the reference genome to distinguish between sequences of two DNA fragments of the first set of the plurality of the cell-free DNA fragments.

17. The method of claim 16 , further comprising performing, by the computer system:

determine an ending or a beginning of a first subsequence of a first DNA fragment of the first set of the plurality of the cell-free DNA fragments based on the two sliding windows aligning to the different regions of the reference genome.

18. The method of claim 16 , wherein the different regions of the reference genome comprise regions on different chromosomes.

19. The method of claim 16 , wherein one or more windows between the two sliding windows are identified as not aligning to the reference genome.

20. The method of claim 19 , further comprising determining the size of a first DNA fragment of the first set of the plurality of the cell-free DNA fragments, wherein determining the size of the first DNA fragment comprises:

determining a length of a longest subsequence that aligns to a single region of the reference genome.

21. The method of claim 1 , wherein the biological sample is plasma or serum.

22. The method of claim 1 , wherein the first set of the cell-free DNA fragments is randomly distributed from a plurality of chromosomes.

23. The method of claim 1 , wherein the first concatemer does not include a spacer fragment having a known sequence between cell-free DNA fragments of the first set of the plurality of the cell-free DNA fragments.

24. The method of claim 1 , wherein the first concatemer does not comprise replicated DNA fragments of the plurality of the cell-free DNA fragments, and the second concatemer does not comprise replicated DNA fragments of the plurality of the cell-free DNA fragments.

25. The method of claim 1 further comprising:

determining a size distribution of the plurality of the cell-free DNA fragments using the subsequences within the first sequence and the subsequences within the second sequence.

26. The method of claim 1 , wherein the biological sample obtained from the single subject is maternal plasma comprising maternal cell-free DNA fragments and fetal cell-free DNA fragments from which the first set of the plurality of the cell-free DNA fragments and the second set of the plurality of the cell-free DNA fragments are obtained.

27. A method comprising:

receiving a biological sample obtained from a single subject, the biological sample including a bodily fluid comprising cell-free DNA fragments;

extracting a plurality of the cell-free DNA fragments from the biological sample;

concatemerizing a first set of the plurality of the cell-free DNA fragments to obtain a first concatemer consisting of the first set of the plurality of the cell-free DNA fragments and optionally spacer DNA fragments, wherein:

the cell-free DNA fragments of the first set have different sequences and are from different locations in the genome of the subject, and

the first concatemer does not comprise replicated DNA fragments of the plurality of the cell-free DNA fragments;

concatemerizing a second set of the plurality of the cell-free DNA fragments to obtain a second concatemer consisting of the second set of the plurality of the cell-free DNA fragments and optionally spacer DNA fragments, wherein the second concatemer comprises a different combination or a different permutation of the cell-free DNA fragments than the first concatemer, wherein:

concatemerizing the second set of the plurality of the cell-free DNA fragments occurs in parallel with the concatemerizing of the first set of the plurality of the cell-free DNA fragments, and

the second concatemer does not comprise replicated DNA fragments of the plurality of the cell-free DNA fragments;

performing single-molecule sequencing of the first concatemer to obtain a first sequence, the first sequence including the different sequences of the first set of the plurality of the cell-free DNA fragments as subsequences within the first sequence; and

performing single-molecule sequencing of the second concatemer to obtain a second sequence, the second sequence including the different sequences of the second set of the plurality of the cell-free DNA fragments as subsequences within the second sequence.