Sample to Sequence

Abstract

Method and sample vessels are provided for amplification and sequencing of nucleic acids in a sample.

1 .- 36 . (canceled) 
     
     
         37 . A container for amplification and sequencing of a plurality of nucleic acids, the container comprising:
 a first-stage amplification chamber, the first-stage amplification chamber comprising a plurality of first-stage pairs of primers, each first-stage pair of primers configured for amplification of one of the plurality of nucleic acids to generate a plurality of first-stage amplicons; and   a second-stage amplification chamber, the second-stage amplification chamber comprising a plurality of second-stage primer pairs, each second-stage primer pairs configured for amplification of at least a portion of a sequence of one of the first stage-amplicons to generate second-stage amplicons,   wherein the second-stage amplification chamber is further configured for sequencing the second-stage amplicons.   
     
     
         38 . The container of  claim 37 , wherein at least one member of each of the second-stage primer pairs is nested within a corresponding first-stage primer. 
     
     
         39 . The container of  claim 37 , wherein at least one member of each of the second-stage primer pairs is tethered to a support. 
     
     
         40 . The container of  claim 39 , wherein the at least one member of each of the second-stage primer pairs is tethered to the support by the 5′ end. 
     
     
         41 . The container of  claim 37 , wherein the second-stage amplicons comprise single molecular species. 
     
     
         42 . The container of  claim 37 , further comprising one or more sealable ports, the one or more sealable ports providing the only access from an exterior of the container to the first-stage amplification chamber and the second-stage amplification chamber, such that when the one or more sealable ports are sealed, the container is fully closed. 
     
     
         43 . The container of  claim 37 , wherein the second-stage amplification chamber comprises an inlet channel and an outlet channel and opening and closing the inlet channel and the outlet channel controls flow of fluid across the second-stage amplification chamber. 
     
     
         44 . The container of  claim 37 , further comprising a lysis chamber configured to receive a sample and to prepare a lysed sample. 
     
     
         45 . The container of  claim 44 , further comprising a nucleic acid extraction chamber configured to receive the lysed sample and to extract nucleic acids from the lysed sample. 
     
     
         46 . The container of  claim 37 , wherein the second-stage amplification chamber comprises an array of wells, each well configured to carry out a second-stage amplification reaction. 
     
     
         47 . The container of  claim 37 , wherein the second-stage amplification chamber comprises an array of spots, each spot comprising one of the second-stage primer pairs, and each spot configured to carry out a second-stage amplification reaction. 
     
     
         48 . The container of  claim 47 , wherein for each spot at least one primer of the second-stage primer pair is tethered to the spot. 
     
     
         49 . The container of  claim 47 , wherein the number of spots totals no more than 512 spots. 
     
     
         50 . The container of  claim 47 , wherein the number of spots totals no more than 256 spots. 
     
     
         51 .- 55 . (canceled) 
     
     
         56 . A container for amplifying and sequencing nucleic acids, the container comprising:
 a first chamber comprising a plurality of first primer pairs, each first primer pair configured to amplify one of the nucleic acids to generate a plurality of first amplicons; and   a second chamber comprising a plurality of second primer pairs, each second primer pair configured to amplify at least a portion of a sequence of one of the first amplicons to generate second amplicons;   wherein the second chamber is further configured for sequencing the second amplicons.   
     
     
         57 . The container of  claim 56 , wherein at least one member of each of the second primer pairs is nested within a corresponding first primer. 
     
     
         58 . The container of  claim 56 , wherein the second amplicons comprise single molecular species. 
     
     
         59 . The container of  claim 56 , further comprising one or more sealable ports providing the only access from an exterior of the container to the first chamber and the second chamber such that when the one or more sealable ports are sealed, the container is fully closed. 
     
     
         60 . The container of  claim 56 , further comprising a lysis chamber configured to receive a sample and to prepare a lysed sample. 
     
     
         61 . The container of  claim 60 , further comprising a nucleic acid extraction chamber configured to receive the lysed sample and to extract nucleic acids from the lysed sample.