The Biological Significance of Multi-copy Regions and Their Impact on Variant Discovery

Identification of genetic variants via high-throughput sequencing(HTS)technologies has been essential for both fundamental and clinical studies.However,to what extent the genome sequence composition affects variant calling remains unclear.In this study,we identified 63,897 multi-copy sequences(MCSs)with a minimum length of 300 bp,each of which occurs at least twice in the human genome.The 151,749 genomic loci(multi-copy regions,or MCRs)harboring these MCSs account for 1.98% of the genome and are distributed unevenly across chromosomes.MCRs containing the same MCS tend to be located on the same chromosome.Gene Ontology(GO)analyses revealed that 3800 genes whose UTRs or exons overlap with MCRs are enriched for Golgirelated cellular component terms and various enzymatic activities in the GO biological function category.MCRs are also enriched for loci that are sensitive to neocarzinostatin-induced double-strand breaks.Moreover,genetic variants discovered by genome-wide association studies and recorded in dbSNP are significantly underrepresented in MCRs.Using simulated HTS datasets,we show that false variant discovery rates are significantly higher in MCRs than in other genomic regions.These results suggest that extra caution must be taken when identifying genetic variants in the MCRs via HTS technologies.

Improving solubility and copy number of taxadiene synthase to enhance the titer of taxadiene in Yarrowia lipolytica

Taxadiene is an important precursor for the biosynthesis of highly effective anticancer drug paclitaxel,but its microbial biosynthesis yield is very low.In this study,we employed Yarrowia lipolytica as a microbial host to produce taxadiene.First,a“push–pull”strategy was adopted to increase taxadiene production by 234%.Then taxadiene synthase was fused with five solubilizing tags respectively,leading a maximum increase of 62.3%in taxadiene production when fused with SUMO.Subsequently,a multi-copy iterative integration method was used to further increase taxadiene titer,achieving the maximum titer of 23.7 mg/L in shake flask culture after three rounds of integration.Finally,the taxadiene titer was increased to 101.4 mg/L by optimization of the fed-batch fermentation conditions.This is the first report of taxadiene biosynthesis accomplished in Y.lipolytica,serving as a good example for the sustainable production of taxadiene and other terpenoids in this oleaginous yeast.

The synergetic effects of two CCAAT boxes in Aspergillus niger glaA gene promoter on activation of PglaA transcription

EMSA and footprinting analyses have revealed that the -489—-414 bp and the -390—-345 bp (designated DC and PC respectively) upstream of the Aspergillus niger T21 glaA gene were bound by one protein factor in the A. niger T21 whole cell extract. Both DC and PC contained CCAAT pentanucleotides. The functions of DC and PC in regulation of expression of glucoamylase (GLA) were studied. CCAAT pentanucleotides were replaced with CGTAA and the mutated DNA fragments DCm and PCm lost the binding activities of protein factors in vitro. In vivo when either DC or PC was mutated or the relative orientations between them were changed on the PglaA, the transcriptional activity of PglaA decreased to a basal level. Introduction of multi-copies of DC into the original site at the PglaA in A. niger T21 decreased the expression of endogenous GLA expression and the exogenous reporter E. coli uidA gene introduced under the PglaA promoter, while having no effect on the uidA gene under the control of PgpdA. EMSA re-vealed that the levels of the specific DNA-binding protein factors in the transformants maintained the same meaning that introduction of multi-copies of DC caused the titration effect. AnghapC gene was cloned from A. niger T21 cDNA and introduced into the DC multi-copied strains. The expression of AnghapC improved the expression of the endogenous GLA and the exogenous gene controlled by PglaA. These results showed that both the CCAAT pentanucleotides were necessary for DC and PC binding to the protein factors, and the simultaneous binding of DC and PC to the protein was necessary for promoting the transcriptional activity of PglaA. AngHapC was the specific positive trans-acting protein factor binding to DC.

Multiplexed site-specific genome engineering in Mycolicibacterium neoaurum by Att/Int system

Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories.For some uncommon microbial hosts,such as Mycolicibacterium and Mycobacterium species,however,it is a challenge.Here,we present a multiplexed integrase-assisted site-specific recombination(miSSR)method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories.First,a single-step multi-copy integration method was established in M.neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy,the efficiencies of which were~100%for no more than three-copy integration events and decreased sharply to~20%for five-copy integration events.Second,the R4,Bxb1 andΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events.Third,a reconstructed mycolicibacterial Xer recombinases(Xer-cise)system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation.As a proof of concept,the biosynthetic pathway of ergothioneine(EGT)in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system.With six copies of the biosynthetic gene clusters(BGCs)of EGT and pentose phosphate isomerase(PRT),the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L,which was 3.77 times of that in the wild strain.The improvements indicated that the miSSR system was an effective,flexible,and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.

A Potential Marker DYF371 for Differentiating Han Population from Non-Han Population in Chinese

DYF371 locus and single‑nucleotide polymorphisms(SNPs)defined as DYF371C or DYF371T were simultaneously examined for 142 Chinese unrelated male individuals,including 90 Han population and 52 minorities.In total,37 SNP-short tandem repeat haplotypes were detected.Nine of these 37 haplotypes were unique(24.32%).The gene diversity and discrimination capacity values are 0.8321 and 0.2606,respectively.Furthermore,only five males(5.55%)in the Han samples were determined that they had one copy of allele with SNP T-type and three copies of alleles with SNP C-types,whereas 14 individuals(26.92%)were observed in minorities’samples.The genotype proportion comprising three distinguishing copies of the allele with SNP C-types in Han sample was greatly lower than that in the sample of minorities.Similar results were shown for allele 11C and 10C.Therefore,the alleles of DYF371 with SNP C-type have the potential to differentiate the Han and non-Han Chinese populations.

Disjoint-Path Routing Mechanism in Mobile Opportunistic Networks

The prevalent multi-copy routing algorithms in mobile opportunistic networks(MONs)easily cause network congestion.This paper introduces a disjoint-path(DP)routing algorithm,where each node can only transmit packets once except the source node,to effectively control the number of packet copies in the network.The discrete continuous time Markov chain(CTMC)was utilized to analyze the state transition between nodes,and the copy numbers of packets with the DP routing algorithm were calculated.Simulation results indicate that DP has a great improvement in terms of packet delivery ratio,average delivery delay,average network overhead,energy and average hop count.