Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations

Abstract

Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq). This method combines an ultra-sensitive single-stranded library construction with barcoding error correction, termed DEEPER-Library; and a DNA capture approach using RNA probes targeting both DNA strands, termed DEEPER-Capture. DEEPER-Seq can create NGS libraries from as little as 20 pg DNA with PCR error correcting capabilities, and capture target sequences at an average ratio of 29.2% by targeting both DNA strands simultaneously with an over 98.6% coverage. Our method tags and sequences each of the two strands of a DNA duplex independently and only scores mutations that are found at the same position in both strands, which allows us to identify mutations with allelic fractions down to 0.03% in a whole exome sequencing (WES) study with a background error rate of one artificial error per 4.8 × 109 nucleotides.

Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers

“…On the other hand, the LTC tcPCR setup and operation are analogous to a standard PCR reaction, and thus are more familiar to a larger number of technicians, and also easier to automate. Additionally, it should be noted that LTC could be paired with any library preparation method that introduces the correct adapter sequences, such as single stranded library prep [23] or transposition [24].…”

Detecting Rare Mutations and DNA Damage with Sequencing-Based Methods

“…The simplest applications of high-fidelity sequencing methods involve random fragmentation and sequencing of entire DNA samples. However, there is growing interest in methods targeting specific regions within the genome such that extreme sequencing depths can be obtained for individual loci, and site-specific mutation frequencies can be quantified down to very low levels [20, 32, 38, 40, 41, 44–48]. Choosing among such methods allows researchers to weigh the tradeoffs between obtaining genome-wide information and achieving maximum possible sampling depth for specific loci of interest.…”

“…Two different general approaches have been applied to target individual loci and still achieve a degree of redundancy by sequencing multiple reads per original DNA copy (Figure 1D). First, many of the library construction methods that we have described thus far have been successfully paired with hybrid capture approaches to highly enrich for specific loci that bind to custom-designed complementary oligonucleotide probes [20, 40, 41, 48]. Second, locus-specific PCR or RCA primers can be used to amplify desired regions.…”

“…This method, known as “duplex sequencing” [28], can distinguish true mutations from repeatable amplification errors resulting from misreading of damaged bases (orange starburst) or replication errors in an early PCR cycle. D) High-fidelity sequencing methods can be targeted to specific loci of interest (purple) by hybridizing prepared library molecules to custom oligonucleotide probes (left) [20, 40, 41, 48] or amplifying with locus-specific primers that build in a UMI to each product. In methods such as “Maximum Depth Sequencing” (MDS) [38], the typical exponential library amplification step is preceded by a linear amplification step to mitigate effects of replication errors in early PCR cycles.…”

Advances in Diagnostic Approaches for Viral Etiologies of Diarrhea: From the Lab to the Field

“…PNAs are synthetic oligonucleotides in which the native sugar-phosphate backbone of DNA is replaced with amino acids, especially N-(2-aminoethyl)-glycine units are linked by amide bonds, and to which purine and pyrimidine bases are attached, giving it the properties both of nucleic acids as well of amino acids (Siddiquee et al, 2015). PNAs are sensitive to detect mutants even in low amounts and performance is next to next-generation deep sequencing (Cai et al, 2016; Wang et al, 2017). PNA-PCR assay is performed to estimate the sensitivity of PNAs on plasmid mixtures comprised of varying amounts of mutant and wild-type plasmids (Hong et al, 2016).…”