Methods for preparing dna reference materials and controls

A control for use in identifying a genotype, comprising a first mixture of nucleic acids and a second mixture of nucleic acids;

• wherein the first mixture of nucleic acids comprises a first plurality of nucleotide sequences, each nucleotide sequence of the plurality encodes a genotype of a first plurality of genotypes, and each genotype of the first plurality of genotypes is associated with a neoplasm;
• the second mixture of nucleic acids comprises a second plurality of nucleotide sequences, and each nucleotide sequence of the second plurality of nucleotide sequences encodes a genotype of a second plurality of genotypes, wherein the second plurality of genotypes comprises genotypes that are not associated with a disease;
• and
• the nucleic acids of the first mixture of nucleic acids or the nucleic acids of the second mixture have an average base pair (bp) length of about 50 bps to about 600 bps.

A DNA library comprising a first mixture of nucleic acids and a second mixture of nucleic acids;

• wherein the first mixture of nucleic acids comprises a first plurality of nucleotide sequences, each nucleotide sequence of the plurality encodes a genotype of a first plurality of genotypes, and each genotype of the first plurality of genotypes is associated with a neoplasm;
• the second mixture of nucleic acids comprises a second plurality of nucleotide sequences, and each nucleotide sequence of the second plurality of nucleotide sequences encodes a genotype of a second plurality of genotypes, wherein the second plurality of genotypes comprises genotypes that are not associated with a disease;
• the nucleic acids of the first mixture of nucleic acids or the nucleic acids of the second mixture have an average base pair (bp) length of about 50 bps to about 600 bps;
• the nucleic acids of the first mixture of nucleic acids or second mixture of nucleic acids comprise one or more adaptors ligated to each end of the nucleic acid, wherein the adaptors comprise a restriction enzyme site, optionally a SapI site, and a non-human sequence, optionally a sequence from a nonintegrating RNA virus, such as a respiratory syncytial virus (RSV);
• and
• wherein the restriction enzyme site is positioned to permit removal of the adaptor from the nucleic acids by the restriction enzyme.

A method of generating a control, comprising:

• (i) obtaining DNA fragments, optionally by fragmenting input DNA;
• (ii) amplifying the DNA fragments by PCR to generate an amplified library;
• and
• (iii) digesting the amplified library with a restriction enzyme to generate a modified library.

A control for use in identifying a genotype, comprising a first mixture of amplified nucleic acids;

• wherein the first mixture of nucleic acids comprises a first plurality of nucleotide sequences, each nucleotide sequence of the plurality encodes a genotype of a first plurality of genotypes, and each genotype of the first plurality of genotypes is associated with a neoplasm;
• the first mixture of nucleic acids is substantially free of methylated DNA;
• and
• the nucleic acids of the first mixture of nucleic acids have an average base pair (bp) length of about 50 bps to about 600 bps.

A control for use in determining the ploidy of a chromosome in a fetus, comprising:

• a first mixture of nucleic acids comprising a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence has sequence homology with the chromosome;
• the second nucleotide sequence has sequence homology with a different chromosome;
• and the ratio of the copy number of the first nucleotide sequence to the copy number of the second nucleotide sequence is greater than 1:1;
• a second mixture of nucleic acids comprising a first nucleotide sequence and a second nucleotide sequence, wherein the ratio of the copy number of the first nucleotide sequence to the copy number of the second nucleotide sequence is 1:1;
• and
• the nucleic acids of the first mixture of nucleic acids or the nucleic acids of the second mixture of nucleic acids comprise amplified cfDNA and have an average base pair (bp) length of about 50 bps to about 600 bps.