Tips for minimizing sample misassignment during multiplexed NGS

NGS multiplexing benefits and challenges

Next generation sequencing (NGS) has emerged as a mainstay for numerous biological applications. With throughput rapidly increasing on commercially available sequencing platforms, scientists are performing higher levels of sample multiplexing as a means to reduce sequencing costs and to take advantage of higher instrument capacity [1]. To accomplish this, individual barcodes (also referred to as sample “indexes" or "indices”), consisting of unique DNA sequences, are added to each sample during library preparation. This enables fragments from each library to be associated with the correct sample during data analysis (a step called “demultiplexing”). The degree of multiplexing has risen greatly in the past few years from 96 samples to 20,000, with the availability of increased number of multiplex barcode sets [2–4].

Pooling indexed samples into one sequencing run, however, comes with an increased risk of sample misassignment. This occurs when sequencing reads are associated with the incorrect samples, a phenomenon also referred to as “sample crosstalk”. Sample crosstalk can be particularly detrimental in highly sensitive applications that require identification of rare sequence variants, such as in low-frequency somatic variant detection when used in cancer research, ancient DNA discovery, and viral detection or microbial profiling [5–9]. While sample misassignment has been acknowledged since sample multiplexing was developed, it has received increased attention recently, as newer, higher throughput sequencing platforms are particularly prone to it.

Novel Unique Dual Index (UDI) adapters mitigate the risk of sample crosstalk

Adapters are a key component of the NGS workflow. Each major sequencing platform has its own recommended adapter strategy, often with multiple options. To select the adapter strategy most suitable for your sequencing project, you need to consider several experimental factors. These include the required level of multiplexing, assay sensitivity, and the often-overlooked manufacturing strategies for the adapters.

Due to the inherent variability and complexity of NGS applications, sample misassignment can come from cross-contamination introduced during oligo manufacturing (discussed below). However, misassignment can also result from issues completely unrelated to manufacturing, such as index hopping that occurs during library preparation or cluster generation, or misread bases within index regions—due to sequencing error or even sample carryover from previous sequencing runs.

As a recognized industry leader in adapter manufacturing, IDT leverages its extensive expertise in custom oligo synthesis and the commitment to its customers to provide high quality adapters, regardless of the NGS instrument, synthesis scale, or quality standard desired. Among our adapter offerings are UDI-containing custom adapters, a set of adapters originating from a collaboration between Illumina and IDT. These adapters contain unique sample index combinations. These specially designed indexes enable users to confidently identify and remove reads that potentially lead to sample misassignment during bioinformatic analysis, thereby avoiding erroneous Interpretation.

xGen Dual Index UMI Adapters provide even higher error correction and sequencing sensitivity by including molecular barcodes, also known as unique molecular identifiers (UMIs).

IDT has presented a series of webinars on the sources of sample misassignment, and how use of UDI-containing adapters can substantially reduce crosstalk errors. Access those recordings here:

• Mitigate index hopping while increasing low-frequency variant detection
• A solution for sample crosstalk and index hopping in multiplexed NGS
• NGS detection of low frequency genetic variants using novel, molecular sequencing adapters

Crosstalk during oligo synthesis can contribute to sample misassignment

One potential contributor to crosstalk among multiplexed samples is oligo cross-contamination introduced during the DNA synthesis of adapters and indexed primers. This source has received far less attention despite a recent report suggesting that crosstalk can be significant, depending on the particular oligo vendor and their manufacturing process [10]. False positives or inconsistent sequencing reads can result from poor-quality adapters or indexed primers with either a low level of purity (a high proportion of non-full-length adapter products) or excessive cross-contaminants (unacceptably high levels of other adapter species).

While low levels of oligo-to-oligo crosstalk often go unnoticed with less-sensitive technologies, the use of cross-contaminated indexed oligos in NGS experiments can contribute to barcode misassignment, which not only confounds highly sensitive research applications, but also introduces uncertainty into the analysis of clinically relevant samples. Traditional purification methods designed to improve full-length content of the desired oligos are, unfortunately, incapable of completely removing cross-contaminants. And without functional testing, it is technically challenging to detect the minute amounts of contamination that may affect assay performance, even with the state-of-art analytical tools.

TruGrade DNA Oligos—ideal for ultra-sensitive NGS applications

Quality requirements for synthetic oligos used in NGS experiments continue to increase in stringency in response to new applications that require greater sensitivity. IDT TruGrade DNA Oligos are synthesized and handled with exclusive IDT proprietary processes that are proven to reduce oligo-to-oligo crosstalk. Yet, at the same time, TruGrade DNA Oligos can be synthesized in a high throughput manufacturing environment to provide quick delivery. This synthesis and processing method effectively minimizes oligo cross-contamination as a contributing factor to barcode misassignment during multiplexed NGS applications.

While dual index adapters enable the detection of misassigned reads during bioinformatic analysis, TruGrade processing leads to a lower number of misassigned reads from the start, which can increase coverage and avoid sequencing capacity waste. Customer-generated NGS sequencing data has demonstrated the superiority of TruGrade DNA Oligos compared to purified oligos from other vendors by minimizing oligo cross contamination, which in turn reduces barcode misassignment by orders of magnitude [10].

TruGrade DNA Oligos are available in both HPLC-purified Custom DNA Oligo and Ultramer^® Oligonucleotide formats, and can be supplied in either 96-well plates or individual tubes. They can also be custom formulated to your required specifications. TruGrade DNA Oligos are recommended for a variety of applications, such as NGS library preparation to incorporate barcoded adapters, PCR when using barcoded fusion primers for multiplexed amplicon sequencing, and in other molecular biology applications sensitive to oligo crosstalk.

Use Table 1 to determine which format (HPLC-purified Custom DNA Oligos or Ultramer Oligonucleotides) is more appropriate for your experiments. In addition to the product types listed below, we also offer a custom formulation/build service to accommodate your specific needs. To inquire about customized orders, please contact our scientific applications support group.

*Typical cross-contamination levels were determined using in-house proprietary QC techniques and feedback provided by our customers.

†Production time depends on the number of oligos ordered. Please inquire for an estimated ship date specific to your order.