PrimerPooler: automated primer pooling to prepare library for targeted sequencing

Abstract

Targeted next-generation sequencing based on PCR amplification involves pooling of hundreds to thousands of primers, for preamplification and subsequent parallel single/multiplex PCR. It is often necessary to allocate the set of primers into subpools, a common issue being potential cross-hybridization. For smaller numbers of primers, pool division can be done manually using trial and error to minimize potential hybridization, but this becomes inefficient and time consuming with increasing numbers of primer pairs. We developed PrimerPooler that automates swapping of primer pairs between any user-defined number of subpools to obtain combinations with low-potential interactions. PrimerPooler performs inter-/intra-primer hybridization analysis to identify the adverse interactions, as well as simultaneous mapping of all primers onto a genome sequence in a single run without requiring a prior index of the genome. This allows detection of overlapping primer pairs and allocation of these primer pairs into separate subpools where tiling approaches are used. Using PrimerPooler, 1153 primer pairs were assigned to three preamplification pools (388, 389 and 376 primer pairs each), then 144 subpools of six- to nine-plex PCR for Fluidigm Access Array PCR, followed by Illumina MiSeq sequencing. With optimized experimental protocols, an average of 3269 reads was achieved for the targeted regions, with 95% of targets covered by at least 50 reads, the minimal depth of reads for confident variant calling. PrimerPooler provides a fast and highly efficient stratification of primer pairs for targeted enrichment, thus ensuring representative amplification of the targeted sequences. PrimerPooler is also able to analyse degenerate primers, and is thus also useful for microbiological identification and related target sequencing.

Variable outcomes of hybridization between declining Alosa alosa and Alosa fallax

“…The alignment was checked for specifically fixed SNP sites, and 48 primer pairs for polymerase chain reaction (PCR) amplifications were initially designed using the Primer3web The 48 primer pairs were tested against A. alosa and A. fallax individuals. Potential interactions between primer pairs and amplicon overlap were examined using PrimerPooler (Brown et al, 2017).…”

Functional and structural basis of extreme non-coding conservation in vertebrate 5’UTRs

“…A total of 384 primer pairs were designed across these h5UTRs using ThermoAlign and following parameters (others are left at default): primer_size_range=16-40, GC_range=25-75, Tm_range=64-72, primer_conc=300, Na=0, K=75, Tris=10, Mg=2, dNTPs=1.2, amplicon_size_min=250, amplicon_size_max=250 110 . Primers were split into 4 pools of 96 pairs using PrimerPooler 111 . A final set of 380 pairs were synthesized by Eurofins Genomics.…”

Multiplex real-time RT-PCR method for the diagnosis of SARS-CoV-2 by targeting viral N2, RdRP and human RP genes

“…Multiplex primer / probe design Programs such as "PrimerPooler", "PrimerPlex" and "Primer3" were used to design multiplex PCR primer and probe sequences that are speci c to viral and human gene targets [38]. In the selection of SARS-CoV-2 primers, attention was paid to the selection of genome regions that differ from other SARS-CoV relatives.…”