Single-molecule sequencing of plasma DNA

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.