Multiplex Preparation of Barcoded Gene Specific DNA Fragments

Abstract

Methods of preparing a plurality of sample-barcoded anchor-domain-flanked gene specific deoxyribonucleic acid (DNA) fragments from a template nucleic acid, e.g., ribonucleic acid (RNA), sample are provided. Aspects of the methods include employing a set of gene specific primer pairs, wherein each pair of gene specific primers is made up of a forward primer and a reverse primer, at least one of which includes a sample barcode domain. The methods find use in a variety of different applications, including high-throughput sequencing, e.g., expression profiling, applications, including of small biological samples, e.g., single-cells.

1 - 63 . (canceled) 
     
     
         64 - 74 . (canceled) 
     
     
         75 . A system comprising:
 nucleic acid amplification device; and   a set of GSPs wherein each pair of GSPs is made up of a forward primer and a reverse primer comprising a sample barcode domain.   
     
     
         76 . The system according to  claim 75 , wherein the forward and reverse primers of each primer pair comprise GSP domains that are experimentally validated as suitable for use in a multiplex amplification assay. 
     
     
         77 . The system according to  claim 75 , wherein the forward and reverse primers are separated by a template distance of 60 to 300 nt. 
     
     
         78 . The system according to  claim 75 , wherein the reverse primers are linked to a solid support. 
     
     
         79 . The system according to  claim 78 , wherein the solid support is a bead. 
     
     
         80 . The system according to  claim 78 , wherein the reverse primers are linked to the solid support by a cleavable linker. 
     
     
         81 . The system according to  claim 78 , wherein the solid support further comprises specific binding pair member. 
     
     
         82 . The system according to  claim 81 , wherein the specific binding pair member specifically binds to a cell surface marker. 
     
     
         83 . The system according to  claim 78 , wherein reverse primers further comprise a solid support barcode domain. 
     
     
         84 . The system according to  claim 78 , wherein the system further comprises a RNA sample. 
     
     
         85 . The system according to  claim 84 , wherein the RNA sample is from a single cell. 
     
     
         86 . The system according to  claim 78 , wherein the device is a thermal cycler. 
     
     
         87 . A kit comprising:
 a set of GSPs wherein each pair of GSPs is made up of a forward primer and a reverse primer at least one of which comprises a sample barcode domain; and   a multi-well plate.   
     
     
         88 . A kit according to  claim 87 , wherein the reverse primers comprise a sample barcode domain. 
     
     
         89 . The kit according to  claim 87 , wherein the forward and reverse primers of each primer pair comprise GSP domains that are experimentally validated as suitable for use in a multiplex amplification assay. 
     
     
         90 . The kit according to  claim 87 , wherein the reverse primers are linked to a solid support. 
     
     
         91 . The kit according to  claim 90 , wherein the solid support is a bead. 
     
     
         92 . The kit according to  claim 91 , wherein the reverse primers are linked to the solid support by a cleavable linker. 
     
     
         93 . The kit according to  claim 87 , wherein the multi-well plate comprises 96 or more wells. 
     
     
         94 . The kit according to  claim 93 , wherein the multi-well plate comprises 384 or more wells. 
     
     
         95 . The kit according to  claim 94 , wherein the multi-well plate comprises 2000 or more wells. 
     
     
         96 . The kit according to  claim 87 , wherein the kit further comprises a reverse transcriptase. 
     
     
         97 . The kit according to  claim 87 , wherein the kit further comprises a DNA polymerase. 
     
     
         98 . The kit according to  claim 97 , wherein the kit further comprises forward and reverse anchor primers. 
     
     
         99 . The kit according to  claim 87 , wherein the kit further comprises a nuclease. 
     
     
         100 . The kit according to  claim 87 , wherein the kit further comprises dNTPs. 
     
     
         101 . The kit according to  claim 87 , wherein the kit further comprises a control.