Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology

Abstract

High-throughput sequencing platforms are increasingly being used for targeted amplicon sequencing because they enable cost-effective sequencing of large sample sets. For meaningful interpretation of targeted amplicon sequencing data and comparison between studies, it is critical that bioinformatic analyses do not introduce artefacts and rely on detailed protocols to ensure that all methods are properly performed and documented. The analysis of large sample sets and the use of predefined indexes create challenges, such as adjusting the sequencing depth across samples and taking sequencing errors or index hopping into account. However, the potential biases these factors introduce to high-throughput amplicon sequencing data sets and how they may be overcome have rarely been addressed. On the example of a nested metabarcoding analysis of 1920 carabid beetle regurgitates to assess plant feeding, we investigated: (i) the variation in sequencing depth of individually tagged samples and the effect of library preparation on the data output; (ii) the influence of sequencing errors within index regions and its consequences for demultiplexing; and (iii) the effect of index hopping. Our results demonstrate that despite library quantification, large variation in read counts and sequencing depth occurred among samples and that the sequencing error rate in bioinformatic software is essential for accurate adapter/primer trimming and demultiplexing. Moreover, setting an index hopping threshold to avoid incorrect assignment of samples is highly recommended.

Optimization of Protocol for Construction of Fungal ITS Amplicon Library for High-Throughput Illumina Sequencing to Study the Mycobiome of Aspen Leaves

“…Index PCR confirmed that the Illumina primer pair had been indexed to the extended PCR products (called multiplexing), which track the samples at the NGS Illumina step [38][39][40]. Initially, we found that index PCR with extended PCR products (ITS + extension) resulted in situations with undesired bands and primer-dimers (Figure 3h, Table 2), which indicated that the extended PCR product had only been partially amplified (perhaps amplifying only fungal primer pairs).…”

Integrating tissue‐direct PCR into genetic identification: An upgraded molecular ecology approach to survey fern gametophytes in the field

“…For cases when all sampled gametophytes are considered for DNA barcoding (e.g., for revealing the community composition of gametophyte populations), we additionally advocate for this TD‐PCR approach. Notably, TD‐PCR can be applied using indexed primers for a multiplexed amplicon (e.g., Guenay‐Greunke et al, 2021 ) using next‐generation sequencing (NGS) (A. Quinlan and L. Y. Kuo et al, unpublished data). When a sampling size is extensive (e.g., >400 samples), costs can be greatly lowered using NGS amplicon sequencing (to <$1 per sample, by our estimation).…”

Best practices in designing, sequencing and identifying random DNA barcodes

“…One redundant design that we found particularly useful is where each 5' inline index is associated with a unique 3' Illumina index and each 3' inline index is associated with a unique 5' Illumina index. Such redundancy can be used to effectively detect primer cross-contamination, "index hopping", and template switching events that can occur during library preparation or on the Illumina flow cell (Illumina, 2017;Guenay-Greunke et al, 2021;Kinsler et al, 2022). These processes generate chimeric sequences, which introduce demultiplexing errors that in turn translate into errors in lineage frequency estimates.…”

“…They cannot expand the multiplexing capacity, but can help detect some index hopping events (those that occur between the Illumina index and the inline index that are on the same primer). The rate of index hopping is much higher on "patterned flow cell" Illumina machines, so we also recommend using a non-patterned flow cell machine for barcode sequencing whenever possible (Illumina, 2017;Guenay-Greunke et al, 2021;Kinsler et al, 2022).…”