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Long insert clone experimental evidence for assembly improvement and chimeric chromosomes detection in an allopentaploid beer yeast.

ABSTRACT: Lager beer is made with the hybrid Saccharomyces pastorianus. Many publicly available S. pastorianus genome assemblies are highly fragmented due to the difficulties of assembling hybrid genomes, such as the presence of homeologous chromosomes from both parental types, and translocations between them. To improve the assembly of a previously sequenced lager yeast hybrid Saccharomyces sp. 790, and elucidate its genome structure, we proposed the use of alternative experimental evidence. We determined the phylogenetic position of Saccharomyces sp. 790 and established it as S. pastorianus 790. Then, we obtained from this yeast a bacterial artificial chromosome (BAC) genomic library with its BAC-end sequences (BESs). To analyze this data, we developed a pipeline (applicable to other assemblies) that classifies BESs pairs alignments according to their orientation. For the case of S. pastorianus 790, paired-end BESs alignments validated parts of the assembly and unpaired-end ones suggested contig joins or misassemblies. Importantly, the BACs library was preserved and used for verification experiments. Unpaired-end alignments were used to upgrade the previous assembly, and provided an improved detection of translocations. With this, we proposed a genome structure of S. pastorianus 790, which was similar to that of other lager yeasts; however, when we estimated chromosome copy number and experimentally measured its genome size, we discovered that one key difference is the outstanding S. pastorianus 790 ploidy level (allopentaploid). Altogether, our results show the value of combining bioinformatic analyses with experimental data such as long-insert clone information to improve a short-read assembly of a hybrid genome.

SUBMITTER: Gomez-Munoz C

PROVIDER: S-EPMC8495930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Synthetic genomic approaches offer unique opportunities to use powerful yeast and Escherichia coli genetic systems to assemble and modify chromosome-sized molecules before returning the modified DNA to the target host. For example, the entire 1 Mb Mycoplasma mycoides chromosome can be stably maintained and manipulated in yeast before being transplanted back into recipient cells. We have previously demonstrated that cloning in yeast of large (>?~?150 kb), high G?+?C (55%) prokaryotic DNA fragments was improved by addition of yeast replication origins every ~100 kb. Conversely, low G?+?C DNA is stable (up to at least 1.8 Mb) without adding supplemental yeast origins. It has not been previously tested whether addition of yeast replication origins similarly improves the yeast-based cloning of large (>150 kb) eukaryotic DNA with moderate G?+?C content. The model diatom Phaeodactylum tricornutum has an average G?+?C content of 48% and a 27.4 Mb genome sequence that has been assembled into chromosome-sized scaffolds making it an ideal test case for assembly and maintenance of eukaryotic chromosomes in yeast. RESULTS:We present a modified chromosome assembly technique in which eukaryotic chromosomes as large as ~500 kb can be assembled from cloned ~100 kb fragments. We used this technique to clone fragments spanning P. tricornutum chromosomes 25 and 26 and to assemble these fragments into single, chromosome-sized molecules. We found that addition of yeast replication origins improved the cloning, assembly, and maintenance of the large chromosomes in yeast. Furthermore, purification of the fragments to be assembled by electroelution greatly increased assembly efficiency. CONCLUSIONS:Entire eukaryotic chromosomes can be successfully cloned, maintained, and manipulated in yeast. These results highlight the improvement in assembly and maintenance afforded by including yeast replication origins in eukaryotic DNA with moderate G?+?C content (48%). They also highlight the increased efficiency of assembly that can be achieved by purifying fragments before assembly.

Project description:BACKGROUND: Clone libraries provide researchers with a powerful resource to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, and allowed the mining of novel enzymes. Libraries are often constructed by cloning large inserts into cosmid or fosmid vectors. Recently, there have been reports of GC bias in fosmid metagenomic libraries, and it was speculated to be a result of fragmentation and loss of AT-rich sequences during cloning. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role. RESULTS: To explore possible mechanisms responsible for sequence bias in clone libraries, we constructed a cosmid library from a human microbiome sample and sequenced DNA from different steps during library construction: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final cosmid library, and we provide evidence that the bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after DNA is introduced into Escherichia coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for promoters and found that rpoD/?(70) promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found the bias to be more correlated with the number of rpoD/?(70) consensus sequences in the genome than with simple GC content. CONCLUSIONS: The GC bias of metagenomic libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/?(70) consensus-like in E. coli may lead to instability, causing loss of the plasmid or loss of the insert DNA that gives rise to the transcription. Despite widespread use of E. coli to propagate foreign DNA in metagenomic libraries, the effects of in vivo transcriptional activity on clone stability are not well understood. Further work is required to tease apart the effects of transcription from those of gene product toxicity.

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Long insert clone experimental evidence for assembly improvement and chimeric chromosomes detection in an allopentaploid beer yeast.

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