Duplex sequencing

Experimental workflow

Duplex sequencing tagged adapters can be used in combination with majority of NGS adapters. In the figures and workflow section of this article Illumina sequencing adapters are used as an example in accordance to the original published protocol.

Adapter annealing

Two oligonucleotides are used for this step (Figure 1: Adapter oligos). One of the oligonucleotides contains 12 nucleotide single stranded random tag sequence followed by a fixed 5 nucleotide sequence (Black sequence in figure 1). In this step oligonucleotides are annealed in a complementary region by incubation at the required temporal condition.

Adapter synthesis

The adapters that annealed successfully are extended and synthesized by a DNA polymerase to complete a double stranded adapter containing complementary tags (Figure 1).

3’-dT-tailing

The extended double stranded adapters are cleaved by HpyCH4III at a specific restriction site located at 3’ side of the tag sequence and will results in a 3’-dT overhang that will be ligated to the 3’-dA overhang on DNA libraries in adapter ligation step (Figure 1).

Library preparation

Double stranded DNA is sheared using one of the methods: Sonication, enzymatic digestion or nebulization. Fragments are size selected using Ampure XP beads. Gel-based size selection is not recommended for this method since it can cause melting of DNA double strands and DNA damage as the results of UV exposure. The size selected fragments of DNA are subjected to 3’-end-dA-tailing.

Adapter ligation

In this step two tagged adapters are ligated from 3’-dT-tails to 3’-dA-tails on both sides of double stranded DNA library fragments. This process results in double stranded library fragments that contain two random tags in each side (Figure 1 and 2). The "DNA:adapter" ratio is crucial in determining the success of ligation.

Insertion of sequencing adapters to tagged libraries

In the last step of duplex sequencing library preparation, Illumina sequencing adapters are added to the tagged double stranded libraries by PCR amplification using primers containing sequencing adapters. During PCR amplification both complementary strands of DNA are amplified and generate two types of PCR products. Product 1 derive from strand 1 which have a unique tag sequence (called α in the figure 2) next to the Illumina adapter 1 and product 2 that have a unique tag (called β in the figure 2) next to illumine adapter 1. The libraries containing duplex tags and Illumina adapters are sequenced using Illumina TruSeq system. Reads that are originating from each single strand of DNA form a group of reads (tag families) that are sharing the same tag. The detected families of reads will be used in next step for analyzing sequencing data.

Efficiency of adapter ligation

Adapter ligation efficiency is very important in a successful duplex sequencing. Extra amount of libraries or adapters can affect the DNA:adapter balance and therefore result in inefficient ligation and excess amount of primer dimers, respectively. Therefore, it is important to keep the molar concentration of DNA:adapter to the optimal ratio that is 0.05.

Tag family size

Efficiency of duplex sequencing depends on final number of DCSs which is directly related to number of reads in each family (family size). If the family size is too small then the DCS can not be assembled and if too many reads sharing the same tag the data yield will be low. Family size is determined by amount of DNA template for PCR amplification and dedicated sequencing lane fraction. The optimal tag family size is between 6 and 12 members. To obtain the optimal family size the amount of DNA template and dedicated sequencing lane fraction need to be adjusted. The following formula take into account the most important variables that can affect depth of coverage (N=40DG÷R) where "N" is number of reads, "D" is desired depth of coverage, "G" is size of DNA target in basepair and "R" is final read length.

Filtering and trimming

Each duplex sequencing read contains a fixed 5-nucleotide sequence (shown in figures in Black color) located upstream of the 12-nucleotide tag sequence. The reads are filter out if they do not have the expected 5-nucleotide sequence or have more than nine identical or ambiguous bases within each tag. The two 12-nucleotide tags at each end of reads are combined and moved to the read header. Two families of reads are formed that originate from two strands of DNA. One family contains reads with αβ header originate from strand 1 and the second family contains reads with βα header originate from strand 2 (Figure 2). Then the reads are trimmed by removing the fixed 5 bp sequence and 4 error prone nucleotides located at the sites of ligation and end repair. The remaining reads are assembled to consensus sequences using single strand consensus sequences (SSCSs) assembly and duplex consensus sequences (DCSs) assembly.

SSCS assembly

Trimmed sequences from the previous step are aligned to the reference genome using Burrows-Wheeler aligner (BWA) and the unmapped reads are removed. The aligned reads that have the same 24 bp tag sequence and genomic region are detected and grouped together (Family αβ and βα in the figure 2). Each group represents a “tag family”. Tag families with lower than three members are removed from the analysis. To remove errors arise during PCR amplification or sequencing, mutations that are supported by less than 70% of the members (reads) are filtered out from the analysis. Then a consensus sequence is generated for each family using the identical sequences in each position of the remaining reads. The consensus sequence is called single strand consensus sequence (SSCS). The SSCS method increases the NGS accuracy to about 20 fold higher, however this method relies on the sequencing information from single strands of DNA and therefore is sensitive to the errors induced at the first round or before PCR amplification.

DCS assembly

The reads from last step are realigned to the reference genome. In this method SSCS family pairs that have complementary tags will be grouped together (Family αβ and βα in figure 2). These reads originate from two complementary strands of DNA. High confidence sequences are selected based on the perfectly matched base calls of each family. The final sequence is called duplex consensus sequence (DCS). True mutations are those that match perfectly between complementary SSCSs. This step filter out remaining errors that raised during first round of PCR amplification or during sample preparation.

Decreasing error rate of sequencing

High error rate (0.01-0.001) of standard NGS platforms that introduced during sample preparation or sequencing is a major limitation for detection of variants present in small fraction of cells. Due to the duplex tagging system and use of information in both strands of DNA, duplex sequencing has significantly decreased the error rate of sequencing about 10 million fold using both SSCS and DCS method.

Increasing accuracy of variant calling

It is challenging to identify rare variants accurately using standard NGS methods with the mutations rate of (10⁻² - 10⁻³). Errors that happen early during sample preparation can be detected as rare variants. An example of such errors is C>A/G>T transversion that is detected in low frequencies using deep sequencing or targeted capture data and arise as the result of DNA oxidation during sample preparation. These types of false positive variants are filter out by duplex sequencing method since mutations need to be accurately matched in both strands of DNA to be validated as true mutations. Duplex sequencing can theoretically detect mutations with frequencies as low as 10⁻⁸ compare to 10⁻² rate of standard NGS methods.

Applicable to majority of NGS platforms

Another advantage of duplex sequencing is that it can be used in combination with majority of NGS platforms without making significant changes to the standard protocols.

Cost

Because duplex sequencing provide a significantly higher sequencing accuracy and uses information in both strands of DNA, this method needs a much higher sequencing depth and therefore is a costly approach. The high cost of duplex sequencing limits its application to targeted and amplicon sequencing at present time and will not be applicable for whole genome sequencing approaches. However, with decreasing cost of NGS, the application of duplex sequencing for larger DNA targets will be more feasible.

Practical application

Duplex sequencing is a new method and its efficiency was studied in limited applications such as detecting point mutations using targeted capture sequencing. More studies need to be performed to expand application and feasibility of duplex sequencing to more complex samples with large number of mutations, indels and copy number variations.

Detection of variants with low frequencies

Duplex sequencing and the significant increase of sequencing accuracy has important impacts on applications such as detection of rare human genetic variants, detection of subclonal mutations involve in mechanisms of resistance to therapy in genetically heterogeneous cancers, screening variants in circulating tumor DNA as a non-invasive biomarker and prenatal screening for detection of genetic abnormalities in fetus.

Copy number detection

Another suggested application for duplex sequencing is detection of DNA/RNA copy numbers by estimating the relative frequency of variants. A method for counting PCR template molecules with application to next-generation sequencing.

Analysis and software

A list of required tools and packages for SSCS and DCS analysis can be found in software package.