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Sequencing de novo Mu transposon insertions across maize tissues

ABSTRACT: We sequenced insertion sites for the maize Mutator (Mu) transposon in leaf, root, endosperm, and pollen from Mu-active plants and Mu-inactive controls. Inherited insertions were identified using matched tissues, and then the abundance of de novo (post-zygotic) insertions was quantified and converted to variant allele frequencies (VAF). This dataset provides insight into the abundance of new transposon insertions within heterogeneous tissues and factors that shape mutation accumulation in multicellular organisms.

ORGANISM(S): Zea mays

PROVIDER: GSE279993 | GEO | 2025/05/09

REPOSITORIES: GEO

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Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors. Solid tumors in mice were generated by somatic transposon mutagenesis with a PiggyBac transposon system. Insertion sites of transposons in 11 tumors and 6 non-cancerous tail controls were determined by Illumina high-throughput sequencing. Insertions were determined both on 5' and 3' sides of the transposon (PB5 and PB3, respectively). Quantitative analysis of read numbers revealed enrichment of certain insertions in tumors, but not in controls, and these enriched insertions identify candidate cancer genes.

Project description:Purpose: The goals of this study are to identify Mutator insertion sites and determine the affect of these insertions on nearby genes Methods: Wiseq-based Mu element profiling was performed as described previously using B73/Mo17 F1 hybrid progeny seedlings. The B73 parent was carried Mutator activity that had been introgressed into the B73 genetic background. The Mo17 parent lacked active Mu elements. Thus, all new insertions were into the B73 genome. Genomic DNA was extracted from 6-day-old seedlings of B73/Mo17 hybrid plants. Amplicon-based enrichment of Mu flanking DNA was then performed. The purified PCR products were subject to Miseq-based Wideseq pipeline at Purdue Genomics Core Facility (https://www.purdue.edu/hla/sites/genomics/wideseq-2/). Wideseq reads were mapped to the B73 reference genome as described previously. By identifying the Mu target site duplications (TSDs), a set of genes targeted by Mu insertions that segregated in hybrid progeny was obtained and those containing B73/Mo17 SNPs in their mRNA sequences were used for allele-specific expression analysis. Because new insertions were into the B73 genome, the effect of these insertions would be expected to be specific in all cases to the B73 allele. To quantify the allele frequency, we performed RT-PCR followed by Wideseq from the identical shoot tissues of the hybrid seedlings mentioned above. Total RNA was extracted using the RNA Extraction Kit (Zymo) and cDNAs were synthesized using Promega M-MLV Reverse Transcriptase. For for a subset of 16 genes that carried SNPs, RNA fragments containing B73/Mo17 SNPs were amplified by RT-PCR. Libraries were prepared using an mRNA preparation kit (Illumina) following a standard protocol (https://www.purdue.edu/hla/sites/genomics/wi-deseq-2/). The RT-PCR products were then sequenced by the “WideSeq” pipeline. Results: A knockdown index was deduced by normalizing the observed ratio to that observed in genes that lacked Mu insertions in both genetic backgrounds for each insertion. We found that none of the four promoter insertions changed the expression of nearby genes. A quarter of 5’ UTR insertions (5 out of 20) caused knockout or strong knockdown effects and one of seven intronic insertions resulted in a knockout effect. Collectively, of a total of 33 Mu insertions, all of which were within 200 bp of genes, only 11 significantly reduced gene expression, and only two knocked it out completely. Conclusions: These results indicate that that Mu element insertions near TSSs of host genes are often associated with quantitative and in many cases neglectable functional consequences on nearby gene expression.

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Sequencing de novo Mu transposon insertions across maize tissues

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