Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix)

Abstract

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

Regional biogeography of microbiota composition in the Chagas disease vector Rhodnius pallescens

“…We amplified bacterial 16S rRNA DNA using the S-D-Bact-0341-b-S-17 (5′-CCT ACG GGN GGC WGC AG-3′) forward and S-D-Bact-0785-a-A-21 (5′-GAC TAC HVG GGT ATC TAA TCC-3′) reverse primer pair [52] to which we added modifications following previous studies [50, 53, 54]. We added Illumina TruSeq sequences to the 5′-end of the forward (Read 1) and reverse (Read 2) primer creating fusion primers.…”

Improved microbial community characterization of 16S rRNA via metagenome hybridization capture enrichment

“…The primer pairs targeting the V3 and V4 16S regions (S-D-Bact-0341-b-S-17 and S-D-Bact-0785a-A-21 ) (Klindworth et al, 2013) were used for amplification of the 16S rRNA gene in rat fecal samples and mock communities; and the primer pair targeting the V4 region (515-F and 806-R) (Caporaso et al, 2012) was used on the mouse fecal samples. We created indexed fusion primers with TruSeq compatible sequencing oligos as previously described using the Adapterama I and This is a provisional file, not the final typeset article Adapterama II systems (Glenn et al, 2019a;Glenn et al, 2019b) to generate amplicon libraries using two rounds of PCR (Method 5 of Table 3 primer, 2.5 µL of 5 µM reverse iTru7 primer, and 5 µL of product from the first PCR. The following were used as PCR conditions: initial denaturation at 95°C for 2 min; 10 cycles of 95°C for 20 sec, 60°C for 15 sec, and 72°C for 30 sec; final extension at 72°C for 5 min.…”

“…Extracted DNA was sheared on a Bioruptor UCD-300 (Diagenode, Denville, NJ, USA) to an average size of about 500 bp. We input ~100 ng of fragmented DNA into each reaction of a KAPA HyperPrep Kit (KAPA Biosystems, Wilmington, MA, USA) following manufacturer's protocol at half volume reaction size with 14 PCR cycles using iTru adaptors and indexed primers (Glenn et al, 2019b). Samples were sequenced on an Illumina HiSeq 3000 with PE150 reads (Oklahoma Medical Research Foundation, Oklahoma City, OK, USA).…”

Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)

“…One of the attractive features of our system is that it separates the primers and stubs into more manageable units. In other Adapterama papers, we use these same iTru primers with different adapter stubs to construct double- to quadruple-indexed amplicon libraries (Glenn et al, 2019), double-digest restriction-site associated DNA (3RAD; Bayona-Vásquez et al, 2019), and RADcap (Hoffberg et al, 2016) libraries. All of these extensions facilitate library preparation, sequencing, and bioinformatic processing of these types of data while also significantly reducing costs.…”

“…The approach allows multiple researchers to have unique primer sets so that libraries from individual researchers can be pooled without worrying about tag overlap. These primers can also be used with a variety of other application-specific adapters described in subsequent Adapterama papers for amplicon and RADseq libraries (Bayona-Vásquez et al, 2019; Glenn et al, 2019; Hoffberg et al, 2016). By modularizing library construction, researchers are free to focus on the development of new application-specific tags.…”