Sequencing methods and compositions for prenatal diagnoses
The invention provides methods for determining aneuploidy and/or fetal fraction in maternal samples comprising fetal and maternal cfDNA by massively parallel sequencing. The method comprises a novel protocol for preparing sequencing libraries that unexpectedly improves the quality of library DNA while expediting the process of analysis of samples for prenatal diagnoses.
1. A method for preparing a sequencing library from a test sample comprising nucleic acid molecules, wherein said nucleic acids molecules are human cell-free DNA (cfDNA) molecules, wherein the method comprises the consecutive steps of end-repairing, dA-tailing and adaptor ligating said nucleic acids molecules, wherein said consecutive steps exclude purifying the end-repaired products prior to the dA-tailing step and exclude purifying the dA-tailing products prior to the adaptor-ligating step, and wherein said consecutive steps are performed in less than 1 hour.
2. The method of claim 1 , wherein said consecutive steps are performed in the absence of polyethylene glycol.
3. The method of claim 1 , wherein said nucleic acids molecules are not subjected to fragmentation prior to the consecutive steps of end-repairing, dA-tailing and adaptor ligating said nucleic acids molecules.
4. The method of claim 1 further comprising sequencing said sequencing library.
5. The method of claim 4 , wherein said sequencing is a next generation sequencing (NGS).
6. The method of claim 4 , wherein said sequencing is massively parallel sequencing.
7. The method of claim 4 , wherein said sequencing comprises an amplification.
8. The method of claim 4 , wherein said sequencing is single molecule sequencing.
9. The method of claim 1 , wherein the sample is a plasma sample derived from peripheral blood that comprises a mixture of cfDNA derived from normal and cancerous cells.
10. The method of claim 6 , wherein:
(i) said sequencing is massively parallel sequencing using sequencing-by-synthesis with reversible dye terminators; or
(ii) said sequencing is massively parallel sequencing using sequencing-by-ligation.