Methods, Systems and Compositions Thereof for Nucleic Acid Library Quality Control and Quantification

Abstract

Methods, systems, kits and compositions are described for quality control and quantitation of nucleic acid libraries of double stranded nucleic acid libraries prior to massively parallel sequencing. Electrophoretic separation within a channel using a detectably labeled single stranded sizing ladder may be used to define the molecular weight range and amount of the double stranded nucleic acids.

1 - 77 . (canceled) 
     
     
         78 . A computer-readable storage medium encoded with instructions, executable by a processor, the instructions including instructions for:
 i. receiving data related to:
 a. migrating a plurality of double stranded nucleic acids labeled with a first dye under mobility dependent separation conditions, wherein the size of each of the double stranded nucleic acids is known, 
 b. migrating a plurality of detectably labeled single stranded reference nucleic acids under the mobility dependent separation conditions, wherein the size of each of the detectably labeled single stranded reference nucleic acids is known, 
 c. separating each of the plurality of double stranded nucleic acids labeled with the first dye and determining a migration time of each of the plurality of double stranded nucleic acids labeled with the first dye, and 
 d. separating each of the detectably labeled single stranded reference nucleic acids and determining a migration time of each of the detectably labeled single stranded reference nucleic acids; 
   ii. interpolating, based on the determined migration times of the plurality of double stranded nucleic acids labeled with the first dye and determined migration times of the detectably labeled single stranded reference nucleic acids, correlated sizes of single stranded reference nucleic acids; and   iii. generating a calibration curve relating double stranded nucleic acids labeled with the first dye and detectably labeled single stranded reference nucleic acids.   
     
     
         79 . A computer-readable storage medium of  claim 78 , further comprising instructions for:
 i. receiving fluorescence data related to a double stranded nucleic acid labeled with a first dye, wherein the fluorescence data includes intensity values over time;   ii. identifying a peak within the fluorescence data;   iii. determining an area of the peak; and   iv. normalizing the area of the peak data to generate a relative concentration value of the double stranded nucleic acid labeled with the first dye.   
     
     
         80 . The computer-readable storage medium of  claim 78 , wherein generating a calibration curve comprises a system comprising:
 i. a fluorescence detector interface configured to receive data related to:
 a. migrating a plurality of double stranded nucleic acids labeled with a first dye under mobility dependent separation conditions, wherein the size of each of the double stranded nucleic acids is known, 
 b. migrating a plurality of detectably labeled single stranded reference nucleic acids under the mobility dependent separation conditions, wherein the size of each of the detectably labeled single stranded reference nucleic acids is known, 
 c. separating each of the plurality of double stranded nucleic acids labeled with the first dye and determining a migration time of each of the plurality of double stranded nucleic acids labeled with the first dye, and 
 d. separating each of the detectably labeled single stranded reference nucleic acids and determining a migration time of each of the detectably labeled single stranded reference nucleic acids; 
   ii. a migration time correlator configured to:
 a. interpolate, based on the determined migration times of the plurality of double stranded nucleic acids labeled with the first dye and determined migration times of the detectably labeled single stranded reference nucleic acids, correlated sizes of single stranded reference nucleic acids, and 
 b. generate a calibration curve relating double stranded nucleic acids labeled with the first dye and detectably labeled single stranded reference nucleic acids. 
   iii. a calibration curve database configured to store the calibration curve, and   iv. a display configured to display the calibration curve to a user.   
     
     
         81 . The computer-readable storage medium of  claim 78 , wherein generating a calibration curve comprises a system comprising:
 i. a processor; and   ii. a memory encoded with instructions, executable by the processor, the instructions for receiving data related to:
 a. migrating a plurality of double stranded nucleic acids labeled with a first dye under mobility dependent separation conditions, wherein the size of each of the double stranded nucleic acids is known, 
 b. migrating a plurality of detectably labeled single stranded reference nucleic acids under the mobility dependent separation conditions, wherein the size of each of the detectably labeled single stranded reference nucleic acids is known, 
 c. separating each of the plurality of double stranded nucleic acids labeled with the first dye and determining a migration time of each of the plurality of double stranded nucleic acids labeled with the first dye, and 
 d. separating each of the detectably labeled single stranded reference nucleic acids and determining a migration time of each of the detectably labeled single stranded reference nucleic acids; 
   iii. interpolating, based on the determined migration times of the plurality of double stranded nucleic acids labeled with the first dye and determined migration times of the detectably labeled single stranded reference nucleic acids, correlated sizes of single stranded reference nucleic acids; and   iv. generating a calibration curve relating double stranded nucleic acids labeled with the first dye and detectably labeled single stranded reference nucleic acids.   
     
     
         82 . A method of determining relative concentration, the method comprising:
 i. receiving fluorescence data related to a double stranded nucleic acid labeled with a first dye, wherein the fluorescence data includes intensity values over time;   ii. identifying a peak within the fluorescence data;   iii. determining an area of the peak; and   iv. normalizing the area of the peak data to generate a relative concentration value of the double stranded nucleic acid labeled with the first dye.   
     
     
         83 . The method of  claim 82 , wherein the method further comprises a system comprising:
 i. a fluorescence detector interface configured to receive fluorescence data related to a double stranded nucleic acid labeled with a first dye, wherein the fluorescence data includes intensity values over time;   ii. a relative concentration calculator configured to:
 a. identify a peak within the fluorescence data, 
 b. determine an area of the peak, and 
 c. normalize the area of the peak data to generate a relative concentration value of the double stranded nucleic acid labeled with the first dye; 
   iii. a relative concentration database configured to store the relative concentration value; and   iv. a display for displaying the relative concentration value to the user.   
     
     
         84 . The method of  claim 82 , wherein the method further comprises a system comprising:
 i. a processor; and   ii. a memory encoded with instructions, executable by the processor, the instructions for:
 a. receiving fluorescence data related to a double stranded nucleic acid labeled with a first dye, wherein the fluorescence data includes intensity values over time; 
 b. identifying a peak within the fluorescence data; 
 c. determining an area of the peak; and 
 d. normalizing the area of the peak data to generate a relative concentration value of the double stranded nucleic acid labeled with the first dye. 
   
     
     
         85 . A system for determining a range of lengths of a plurality of double stranded nucleic acids; comprising:
 i. a reactor vessel configured for
 a. contacting a first dye with a plurality of double stranded nucleic acids, wherein the first dye labels each of the plurality of double stranded nucleic acids, and 
 b. permitting addition of a detectably labeled single stranded sizing ladder; 
   ii. a channel configured to migrate a mixture of the detectably labeled single stranded sizing ladder and a plurality of first dye-labeled double stranded nucleic acids;   iii. an anode and a cathode operably connected to the channel, configured to provide an electric field to the channel; and   iv. a detector configured to detect the first dye and a label of the detectably labeled single stranded sizing ladder.   
     
     
         86 . The system of  claim 85 , wherein the detector detects uv absorbance, visible light absorbance, fluorescence, or chemiluminescence. 
     
     
         87 . The system of  claim 85 , further comprising a temperature controllable environment for the channel. 
     
     
         88 . The system of  claim 85 , further comprising a temperature controllable environment for the reactor vessel. 
     
     
         89 . The system of  claim 85 , further comprising a data processor operably connected to the detector. 
     
     
         90 . The system of  claim 85 , wherein the channel further comprises a separation medium. 
     
     
         91 . The system of  claim 90 , wherein the separation medium is a sieving medium. 
     
     
         92 . The system of  claim 85 , wherein the channel further comprises a denaturing additive. 
     
     
         93 . The system of  claim 85 , wherein the channel is configured within a microfluidic chip. 
     
     
         94 . The system of  claim 85 , wherein the channel is an electrophoretic capillary. 
     
     
         95 . The system of  claim 85 , wherein the channel is part of a multichannel array. 
     
     
         96 . The system of  claim 85 , further comprising a kit for determining a range of lengths of a plurality of double stranded nucleic acids; comprising:
 i. a first dye configured to label a double stranded nucleic acid; and   ii. a detectably labeled single stranded sizing ladder.   
     
     
         97 . The system of  claim 85 , further comprising a composition for determining a range of lengths of a plurality of double stranded nucleic acids; comprising:
 iii. a first dye configured to label a double stranded nucleic acid;   iv. a plurality of double stranded nucleic acids; and   v. a detectably labeled single stranded sizing ladder.