Target-enriched multiplexed parallel analysis for assessment of fetal dna samples

Abstract

The invention provides methods for assessment of fetal DNA samples using target-enriched multiplexed parallel analysis. The methods of the invention utilize Target Capture Sequences (TACS) to thereby enrich for target sequences of interest, followed by massive parallel sequencing and statistical analysis of the enriched population. The methods can be used with fetal or embryonic DNA samples, for example for the detection of the presence of genetic abnormalities, e.g., for purposes of IVF Pre-implantation Genetic Screening (PGS) and Diagnosis (PGD). Kits for carrying out the methods of the invention are also provided.

1 . A kit comprising a container comprising a pool of TArget Capture Sequences (TACS), wherein the pool of TACS comprises a plurality of TACS families,
 wherein each TACS family comprises a plurality of member sequences,   wherein each member sequence binds to the same genomic sequence of interest but has different start and/or stop positions with respect to a reference coordinate system for the genomic sequence of interest,   and further wherein:   (i) each member sequence within each TACS family is between 100-500 base pairs in length, each member sequence having a 5′ end and a 3′ end;   (ii) each member sequence binds to the same genomic sequence of interest at least 50 base pairs away, on both the 5′ end and the 3′ end, from regions harboring Copy Number Variations (CNVs), Segmental duplications or repetitive DNA elements; and   (iii) the GC content of the pool of TACS is between 19% and 80%, as determined by calculating the GC content of each member within each family of TACS.   
     
     
         2 . The kit according to  claim 1 , wherein the start and/or stop positions of the member sequences are staggered by at least 3 base pairs. 
     
     
         3 . The kit according to  claim 1 , wherein the pool of TACS comprises at least 2 TACS families. 
     
     
         4 . The kit according to  claim 1 , wherein the pool of TACS comprises at least 50 different TACS families. 
     
     
         5 . The kit according to  claim 1 , wherein each TACS family comprises at least 2 member sequences. 
     
     
         6 . The kit according to  claim 1 , wherein the pool of TACS is fixed to a solid support. 
     
     
         7 . The kit according to  claim 6 , wherein the pool of TACS are biotinylated and are bound to streptavidin-coated magnetic beads. 
     
     
         8 . The kit according to  claim 1 , wherein the binding of the member sequences encompasses all chromosomes within the human genome. 
     
     
         9 . The kit according to  claim 1 , wherein the member sequences bind to one or more genomic sequences of interest comprising a genetic abnormality. 
     
     
         10 . The kit according to  claim 1 , wherein the start and/or stop positions for the member sequences within a TACS family are listed in table 1. 
     
     
         11 . The kit according to  claim 1 , wherein the different member sequences have different % GC content. 
     
     
         12 . The kit according to  claim 1 , wherein the kit is for use in IVF Pre-implantation Genetic Screening (PGS) and Diagnosis (PGD). 
     
     
         13 . The kit according to  claim 1 , wherein the kit further comprises one or more of the following:
 (i) one or more components for isolating cell free DNA from a biological sample;   (ii) one or more components for preparing the sequencing library comprising primers, adapters, buffers, linkers, restriction enzymes, ligation enzymes and polymerase enzymes;   (iii) one or more components for amplifying and/or sequencing the enriched library; and/or   (iv) software for performing statistical analysis.