Methods of designing a polynucleotide sequence for expressing a polypeptide-of-interest in a cell are provided. Also provided are artificial transcript sequences generated according to the present teachings. Further provided are methods of estimating the adaptiveness of a transcript sequence encoding a polypeptide-of-interest to a gene expression machinery in a cell.

Methods of designing a polynucleotide sequence for expressing a polypeptide-of-interest in a cell are provided. Also provided are artificial transcript sequences generated according to the present teachings. Further provided are methods of estimating the adaptiveness of a transcript sequence encoding a polypeptide-of-interest to a gene expression machinery in a cell.

Provided herein is a method for sequence analysis that comprises analyzing PCR reactions that each contain different portions of the same sample, wherein at least some of the primer pairs are in more than one PCR reaction and at least one of the PCR reactions contains some but not all of the primer pairs of the other reaction(s).

Methods and systems for single molecule sequencing using concatemers of copies of sense and antisense strands. Concatemers are provided, for example, by carrying out rolling circle amplification on a circular molecule having sense and antisense regions to produce repeated copies of the sense and antisense regions connected by linking regions. The circular molecules can be produced by ligating hairpin adapters to each end of a double-stranded nucleic acid having a sense and antisense strand. The ligations can be carried out, for example using blunt end ligation. In some cases, a single molecule consensus sequence for a single template molecule is obtained. A single read from each template molecule can be obtained by comparing the sequence information of the sense and antisense regions.

Methods and systems for single molecule sequencing using concatemers of copies of sense and antisense strands. Concatemers are provided, for example, by carrying out rolling circle amplification on a circular molecule having sense and antisense regions to produce repeated copies of the sense and antisense regions connected by linking regions. The circular molecules can be produced by ligating hairpin adapters to each end of a double-stranded nucleic acid having a sense and antisense strand. The ligations can be carried out, for example using blunt end ligation. In some cases, a single molecule consensus sequence for a single template molecule is obtained. A single read from each template molecule can be obtained by comparing the sequence information of the sense and antisense regions.

Aspects of the invention relate to methods, compositions and algorithms for designing and producing a target nucleic acid. The method can include: (1) providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends thereof; (2) producing via enzymatic digestion a plurality of cohesive-end double-stranded nucleic acid fragments each having two different and non-complementary overhangs; (3) ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase; and (4) forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid. In certain embodiments, the plurality of blunt-end double-stranded nucleic acid fragments can be provided by: releasing a plurality of oligonucleotides synthesized on a solid support; and synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.

Aspects of the invention relate to methods, compositions and algorithms for designing and producing a target nucleic acid. The method can include: (1) providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends thereof; (2) producing via enzymatic digestion a plurality of cohesive-end double-stranded nucleic acid fragments each having two different and non-complementary overhangs; (3) ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase; and (4) forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid. In certain embodiments, the plurality of blunt-end double-stranded nucleic acid fragments can be provided by: releasing a plurality of oligonucleotides synthesized on a solid support; and synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.

The invention provides systems and methods for analyzing viruses by representing viral genetic diversity with a directed acyclic graph (DAG), which allows genetic sequencing technology to detect rare variations and represent otherwise difficult-to-document diversity within a sample. Additionally, a host-specific sequence DAG can be used to effectively segregate viral nucleic acid sequence reads from host sequence reads when a sample from a host is subject to sequencing. Known viral genomes can be represented using a viral reference DAG and the viral sequence reads from the sample can be compared to viral DAG to identify viral species or strains from which the reads were derived. Where the viral sequence reads indicate great genetic diversity in the virus that was infecting the host, those reads can be assembled into a DAG that itself properly represents that diversity.

The invention provides systems and methods for analyzing viruses by representing viral genetic diversity with a directed acyclic graph (DAG), which allows genetic sequencing technology to detect rare variations and represent otherwise difficult-to-document diversity within a sample. Additionally, a host-specific sequence DAG can be used to effectively segregate viral nucleic acid sequence reads from host sequence reads when a sample from a host is subject to sequencing. Known viral genomes can be represented using a viral reference DAG and the viral sequence reads from the sample can be compared to viral DAG to identify viral species or strains from which the reads were derived. Where the viral sequence reads indicate great genetic diversity in the virus that was infecting the host, those reads can be assembled into a DAG that itself properly represents that diversity.

Techniques for generating therapy biomarker scores and visualizing same. The techniques include determining, using a patient's sequence data and distributions of biomarker values across one or more reference populations, a first set of normalized scores for a first set of biomarkers associated with a first therapy, and a second set of normalized scores for a second set of biomarkers associated with a second therapy, generating a graphical user interface (GUI) including a first portion associated with the first therapy and having at least one visual characteristic determined based on a normalized score of the respective biomarker in the first set of normalized scores; and a second portion associated with a second therapy and having at least one visual characteristic determined based on a normalized score of the respective biomarker in the second set of normalized scores; and displaying the generated GUI.