Embodiments provided herewith are directed to self-assembled methods of preparing a patterned surface for sequencing applications including, for example, a patterned flow cell or a patterned surface for digital fluidic devices. The methods utilize photolithography to create a patterned surface with a plurality of microscale or nanoscale contours, separated by hydrophobic interstitial regions, without the need of oxygen plasma treatment during the photolithography process. In addition, the methods avoid the use of any chemical or mechanical polishing steps after the deposition of a gel material to the contours.

Embodiments provided herewith are directed to self-assembled methods of preparing a patterned surface for sequencing applications including, for example, a patterned flow cell or a patterned surface for digital fluidic devices. The methods utilize photolithography to create a patterned surface with a plurality of microscale or nanoscale contours, separated by hydrophobic interstitial regions, without the need of oxygen plasma treatment during the photolithography process. In addition, the methods avoid the use of any chemical or mechanical polishing steps after the deposition of a gel material to the contours.

The present invention belongs to the field of diagnosis of disease. Thus the present invention is focused on a method and kit and system for quantifying the level of minimal residual disease (MRD) in a subject who has been treated for said disease, as well as a method of treatment of said disease in a subject which comprises a step of quantifying the level of minimal residual diseases, wherein said quantifying comprises: (a) identifying, amplifying and sequencing a nucleotide sequence in a biological sample obtained from said subject after treatment for said disease, wherein the gDNA of said biological sample has an average weight, k, per cell, and wherein said nucleotide sequence is identified using primers and is amplified using an amount, D, to afford a first list of characters; (b) identifying, amplifying and sequencing a nucleotide sequence in a biological sample obtained from a subject with said disease using the same primers as in step (a) to afford a second list of characters; (c) determining, for each first list of characters obtained in step (a), the degree of similarity, DS, with each second list of characters obtained in step (b); (d) selecting, for each first list of characters obtained in step (a), the DS of highest value, DS.sub.HV; (e) adding up the number of first lists of characters obtained in step (a) which have a DS.sub.HV that is greater than a threshold value, T, to obtain L.sub.c; (f) adding up the total number of lists of characters, L.sub.t, in the first list of characters; and (g) calculating the level of minimal residual disease (MRD) according to either of the following formulae:
MRD=(L.sub.c×k)/(L.sub.t×D)
or
MRD=L.sub.c/L.sub.t
or
MRD=L.sub.c×(D/k)/L.sub.t.sup.2.

The present invention belongs to the field of diagnosis of disease. Thus the present invention is focused on a method and kit and system for quantifying the level of minimal residual disease (MRD) in a subject who has been treated for said disease, as well as a method of treatment of said disease in a subject which comprises a step of quantifying the level of minimal residual diseases, wherein said quantifying comprises: (a) identifying, amplifying and sequencing a nucleotide sequence in a biological sample obtained from said subject after treatment for said disease, wherein the gDNA of said biological sample has an average weight, k, per cell, and wherein said nucleotide sequence is identified using primers and is amplified using an amount, D, to afford a first list of characters; (b) identifying, amplifying and sequencing a nucleotide sequence in a biological sample obtained from a subject with said disease using the same primers as in step (a) to afford a second list of characters; (c) determining, for each first list of characters obtained in step (a), the degree of similarity, DS, with each second list of characters obtained in step (b); (d) selecting, for each first list of characters obtained in step (a), the DS of highest value, DS.sub.HV; (e) adding up the number of first lists of characters obtained in step (a) which have a DS.sub.HV that is greater than a threshold value, T, to obtain L.sub.c; (f) adding up the total number of lists of characters, L.sub.t, in the first list of characters; and (g) calculating the level of minimal residual disease (MRD) according to either of the following formulae:
MRD=(L.sub.c×k)/(L.sub.t×D)
or
MRD=L.sub.c/L.sub.t
or
MRD=L.sub.c×(D/k)/L.sub.t.sup.2.

Provided herein are systems, methods, and articles of manufacture for collecting and merging two different size droplets using a substrate comprising a plurality of trapping sites. In certain embodiments, provided herein are systems composed of a plurality of larger droplets and smaller droplets and a substrate comprising a plurality of trapping sites where each trapping site is configured to trap only one of the larger droplets and only one of the smaller droplets when the larger droplet is already present at the trapping site. In particular embodiments, the larger and/or smaller droplets are sorted prior to being contacted with the substrate to ensure they contain the desired component (e.g., cell or barcoded bead). In other embodiments, each trapping site is composed of one or multiple fluidically linked capture wells. In some embodiments, collected larger and smaller droplets are merged (e.g., via a demulsifier or electricity).

Provided herein are systems, methods, and articles of manufacture for collecting and merging two different size droplets using a substrate comprising a plurality of trapping sites. In certain embodiments, provided herein are systems composed of a plurality of larger droplets and smaller droplets and a substrate comprising a plurality of trapping sites where each trapping site is configured to trap only one of the larger droplets and only one of the smaller droplets when the larger droplet is already present at the trapping site. In particular embodiments, the larger and/or smaller droplets are sorted prior to being contacted with the substrate to ensure they contain the desired component (e.g., cell or barcoded bead). In other embodiments, each trapping site is composed of one or multiple fluidically linked capture wells. In some embodiments, collected larger and smaller droplets are merged (e.g., via a demulsifier or electricity).

This disclosure provides systems and methods for sample processing and data analysis. Sample processing may include nucleic acid sample processing and subsequent sequencing. Some or all of a nucleic acid sample may be sequenced to provide sequence information, which may be stored or otherwise maintained in an electronic storage location. The sequence information may be analyzed with the aid of a computer processor, and the analyzed sequence information may be stored in an electronic storage location that may include a pool or collection of sequence information and analyzed sequence information generated from the nucleic acid sample. Methods and systems of the present disclosure can be used, for example, for the analysis of a nucleic acid sample, for producing one or more libraries, and for producing biomedical reports. Methods and systems of the disclosure can aid in the diagnosis, monitoring, treatment, and prevention of one or more diseases and conditions.

This disclosure provides systems and methods for sample processing and data analysis. Sample processing may include nucleic acid sample processing and subsequent sequencing. Some or all of a nucleic acid sample may be sequenced to provide sequence information, which may be stored or otherwise maintained in an electronic storage location. The sequence information may be analyzed with the aid of a computer processor, and the analyzed sequence information may be stored in an electronic storage location that may include a pool or collection of sequence information and analyzed sequence information generated from the nucleic acid sample. Methods and systems of the present disclosure can be used, for example, for the analysis of a nucleic acid sample, for producing one or more libraries, and for producing biomedical reports. Methods and systems of the disclosure can aid in the diagnosis, monitoring, treatment, and prevention of one or more diseases and conditions.

In one embodiment, provided are a method for analyzing at least one nucleic acid that can conveniently and highly accurately analyze even a very small number of analyte at least one nucleic acid. In one embodiment, the present invention relates to a method for analyzing at least one nucleic acid, comprising: a library preparation step of preparing a library comprising at least one standard nucleic acid of specific copy number(s) and at least one analyte nucleic acid in a same system; a calibration curve data generation step of generating calibration curve data based on the copy number(s) of the at least one standard nucleic acid of specific copy number(s); and an analyte nucleic acid analysis step of identifying at least one nucleotide sequence of the analyte nucleic acid while identifying the number(s) of the at least one nucleotide sequence of the at least one analyte nucleic acid using the calibration curve data.

In one embodiment, provided are a method for analyzing at least one nucleic acid that can conveniently and highly accurately analyze even a very small number of analyte at least one nucleic acid. In one embodiment, the present invention relates to a method for analyzing at least one nucleic acid, comprising: a library preparation step of preparing a library comprising at least one standard nucleic acid of specific copy number(s) and at least one analyte nucleic acid in a same system; a calibration curve data generation step of generating calibration curve data based on the copy number(s) of the at least one standard nucleic acid of specific copy number(s); and an analyte nucleic acid analysis step of identifying at least one nucleotide sequence of the analyte nucleic acid while identifying the number(s) of the at least one nucleotide sequence of the at least one analyte nucleic acid using the calibration curve data.