Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population

Abstract

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next-generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty-five genes belonging to carotenoids and folate metabolism were PCR-amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600-bp amplicons were directly sequenced in a non-overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128-fold pooling. An evaluation of six different software programs (camba, crisp, gatk unified genotyper, lofreq, snver and vipr) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.

Identification of phytic acid mutants in oilseed rape (Brassica napus) by large‐scale screening of mutant populations through amplicon sequencing

“…Today, only a few bespoke bioinformatic pipelines are available for the identification of mutations in polyploids, namely M aps (Henry et al , ) and C amba (Missirian et al , ; Tsai et al , ). A recent study in tomato ( Solanum lycopersicum ) discussed all the existing bioinformatics pipelines for reducing the occurrence of false positives (Gupta et al , ). The authors of that study claimed the need for establishing robust pipelines for studies involving multidimensional pooling and amplicon stacking.…”

Development and Characterization of an Ethyl Methane Sulfonate (EMS) Induced Mutant Population in Capsicum annuum L.

“…Advanced NGS platforms like Illumina, SOL-iD, Ion Torrent, and PacBio and the steady reduction in sequencing costs has also contributed to the combination of NGS and TILLING methods. This approach can now be used efficiently in grains and horticultural crops [54][55][56]. Hence, the mutant population developed here can be analyzed by TILLING and sequencing, allowing the fast and robust detection of mutant alleles for use in pepper breeding in the near future.…”

Fragmentation of pooled PCR products for highly multiplexed TILLING

“…Barley genomic DNA used in this study were previously prepared for traditional TILLING assays 15 . Samples were pooled in a three dimensional array as previously described except that pooling was higher, with 256 unique mutant lines in each dimension 3,16 . PCR amplification using previously validated primers (Table S3) was performed as previously described 30 .…”

“…All samples were adjusted to a concentration of 200 ng. PCR products were pooled together according to the three dimensional pooling scheme 16 . Sequencing libraries were prepared using TruSeq Nano DNA HT Library Prep Kit (Illumina, Catalogue # FC-121-9010DOC) with 200 ng of starting pooled PCR products according to manufacturer's recommendations.…”

“…Base qualities were prepared using pysamstats and the baseq feature (https://github.com/alimanfoo/pysamstats). Screening of data for previously identified SNP mutations was performed using the CAMBa pipeline for three dimensionally pooled samples as previously described 3,16 .…”