Abstract: Methods and compositions for making DNA libraries for massive parallel next generation sequencing (NGS), comprises two parts. These methods may be referred to as Triseq sequencing. The first part includes ligating a UMI adapter, amplifying the DNA fragments in the presence of dUTP, enriching the target molecules through primer extension by using a panel of both forward and reverse primers, and removing the dU-containing template DNA. The DNA molecules are organized to primary clones and subclones, labeled by the UMI on 5? and 3? end of the DNA fragments, respectively. The second part includes sequencing the DNA library by NGS, deducing consensus sequence from each subclone, and from within each primary clone, and between the consensus sequences obtained from both forward and reverse primers.

Abstract: A method for preparing a homopolymer recalibration panel includes: extracting, from a set of amplicons used in sequencing-by-synthesis, a set of candidate amplicons satisfying a first set of criteria, wherein the first set of criteria includes amplicons known to belong to high-confidence regions of a reference genome with no variants; and selecting, from the set of candidate amplicons, a reduced set of amplicons satisfying a second set of criteria, wherein the second set of criteria includes amplicons that together comprise at least a minimal threshold number of homopolymers of each homopolymer length between a predetermined minimal homopolymer length and a predetermined maximal homopolymer length for one or more of homopolymer types A, T, C, and G.

Abstract: Methods, apparatuses and compositions for generating highly sensitive and accurate sequencing results of massive parallel sequencing (NGS). The methods and compositions may be referred to as SubDivision-Seq, and may comprise two parts. The first part includes making a target-enriched DNA library, organizing the UMIs on DNA molecules to form primary clones and subdividing the primary clones into subclones. The second part includes sequencing the DNA library by NGS, deducing consensus sequence from each subclone, then deducing consensus sequence in each primary clone.

Abstract: A method for preparing a homopolymer recalibration panel includes: extracting, from a set of amplicons used in sequencing-by-synthesis, a set of candidate amplicons satisfying a first set of criteria, wherein the first set of criteria includes amplicons known to belong to high-confidence regions of a reference genome with no variants; and selecting, from the set of candidate amplicons, a reduced set of amplicons satisfying a second set of criteria, wherein the second set of criteria includes amplicons that together comprise at least a minimal threshold number of homopolymers of each homopolymer length between a predetermined minimal homopolymer length and a predetermined maximal homopolymer length for one or more of homopolymer types A, T, C, and G.