Ultra-accurate microbial amplicon sequencing with synthetic long reads.

BACKGROUND: Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. METHODS: Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. RESULTS: LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. CONCLUSIONS: The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics. Video abstract.

Targeted transcriptome analysis using synthetic long read sequencing uncovers isoform reprograming in the progression of colon cancer.

The characterization of human gene expression is limited by short read lengths, high error rates and large input requirements. Here, we used a synthetic long read (SLR) sequencing approach, LoopSeq, to generate accurate sequencing reads that span full length transcripts using standard short read data. LoopSeq identified isoforms from control samples with 99.4% accuracy and a 0.01% per-base error rate, exceeding the accuracy reported for other long-read technologies. Applied to targeted transcriptome sequencing from colon cancers and their metastatic counterparts, LoopSeq revealed large scale isoform redistributions from benign colon mucosa to primary colon cancer and metastatic cancer and identified several previously unknown fusion isoforms. Strikingly, single nucleotide variants (SNVs) occurred dominantly in specific isoforms and some SNVs underwent isoform switching in cancer progression. The ability to use short reads to generate accurate long-read data as the raw unit of information holds promise as a widely accessible approach in transcriptome sequencing.

Satb1 ablation alters temporal expression of immediate early genes and reduces dendritic spine density during postnatal brain development.

Complex behaviors, such as learning and memory, are associated with rapid changes in gene expression of neurons and subsequent formation of new synaptic connections. However, how external signals are processed to drive specific changes in gene expression is largely unknown. We found that the genome organizer protein Satb1 is highly expressed in mature neurons, primarily in the cerebral cortex, dentate hilus, and amygdala. In Satb1-null mice, cortical layer morphology was normal. However, in postnatal Satb1-null cortical pyramidal neurons, we found a substantial decrease in the density of dendritic spines, which play critical roles in synaptic transmission and plasticity. Further, we found that in the cerebral cortex, Satb1 binds to genomic loci of multiple immediate early genes (IEGs) (Fos, Fosb, Egr1, Egr2, Arc, and Bdnf) and other key neuronal genes, many of which have been implicated in synaptic plasticity. Loss of Satb1 resulted in greatly alters timing and expression levels of these IEGs during early postnatal cerebral cortical development and also upon stimulation in cortical organotypic cultures. These data indicate that Satb1 is required for proper temporal dynamics of IEG expression. Based on these findings, we propose that Satb1 plays a critical role in cortical neurons to facilitate neuronal plasticity.

Multivariate analysis of a 3D mass spectral image for examining tissue heterogeneity.

The tissue microenvironment critically influences the molecular characteristics of a tumor. However, as tumorous tissue is highly heterogeneous it may harbor various sub-populations with different microenvironments, greatly complicating the unambiguous analysis of tumor biology. Mass spectrometry imaging techniques allow for the direct analysis of tumors in the spatial context of their microenvironment. However, discovery of heterogeneous sub-populations often depends on the use of multivariate statistical methods. While this is routinely used for 2D images, multivariate statistical approaches are rarely seen in the context of 3D images. Here we present the automatic alignment of 2D images recorded by nanostructure-initiator mass spectrometry (NIMS) to reconstruct a 3D model of a mouse mammary tumor. Multivariate statistical analysis was applied to the whole 3D reconstruction at once, revealing distinct tumor regions, an observation that would not have been possible in such clarity through the analysis of isolated 2D sections. These sub-structures were confirmed by H&E and Oil Red O stains. This study shows that the combination of 3D imaging and multivariate statistics can be used to define tumor regions.

Complexity in transcription control at the activation domain-mediator interface.

Transcript elongation by polymerase II paused at the Egr1 promoter is activated by mitogen-activated protein kinase phosphorylation of the ternary complex factor (TCF) ELK1 bound at multiple upstream sites and subsequent phospho-ELK1 interaction with mediator through the MED23 subunit. Consequently, Med23 knockout (KO) nearly eliminates Egr1 (early growth response factor 1) transcription in embryonic stem (ES) cells, leaving a paused polymerase at the promoter. Med23 KO did not, however, eliminate Egr1 transcription in fibroblasts. Chromatin immunoprecipitation analysis and direct visualization of fluorescently labeled TCF derivatives and mediator subunits revealed that three closely related TCFs bound to the same control regions. The relative amounts of these TCFs, which responded differently to the loss of MED23, differed in ES cells and fibroblasts. Transcriptome analysis suggests that most genes expressed in both cell types, such as Egr1, are regulated by alternative transcription factors in the two cell types that respond differently to the same signal transduction pathways.

Genetic selection system for improving recombinant membrane protein expression in E. coli.

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold.

Mediator requirement for both recruitment and postrecruitment steps in transcription initiation.

Mediator complexes are required for activators to stimulate Pol II preinitiation complex assembly on an associated promoter. We show here that for the mouse Egr1 gene, controlled largely by MAP kinase phosphorylation of the ELK1 transcription factor, the MED23 Mediator subunit that interacts with phospho-ELK1 is also required to stimulate Pol II initiation at a step subsequent to preinitiation complex assembly. In Med23-/- cells, histone acetylation, methylation, and chromatin remodeling complex association at the Egr1 promoter were equivalent to that of wild-type cells, yet Egr1 induction was greatly reduced. MAP kinase activation stimulated Pol II and GTF promoter binding. However, the difference in factor binding between wild-type and mutant cells was much less than the difference in transcription, and Pol II remained localized to the promoter in mutant cells. These results indicate that an interaction with MED23 stimulates initiation by promoter bound Pol II in addition to Pol II and GTF recruitment.

Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors.

For gene therapy of inherited diseases, targeted integration/gene repair through homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to avoid potentially serious problems of random integration such as cellular transformation and gene silencing. Efficient sequence-specific modification of chromosomes by HR would also advance both biological studies and therapeutic applications of a variety of stem cells. Toward these goals, we developed an improved strategy of adenoviral vector (AdV)-mediated HR and examined its ability to correct an insertional mutation in the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse ES cells. The efficiency of HR was compared between four types of AdVs that contained various lengths of homologies at the Hprt locus and with various multiplicities of infections. The frequency of HR with helper-dependent AdVs (HD AdVs) with an 18.6-kb homology reached 0.2% per transduced cell at a multiplicity of infection of 10 genomes per cell. Detection of random integration at DNA levels by PCR revealed extremely high efficiency of 5% per cell. We also isolated and characterized chromosomal sites where HD AdVs integrated in a random manner. In contrast to retroviral, lentiviral, and adeno-associated viral vectors, which tend to integrate into genes, the integration sites of AdV was distributed randomly inside and outside genes. These findings suggest that HR mediated by HD AdVs is efficient and relatively safe and might be a new viable option for ex vivo gene therapy as well as a tool for chromosomal manipulation of a variety of stem cells.

Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector.

Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Delta) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected approximately 50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Delta Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Delta Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism.