Patient-derived organoids (PDOs) have recently emerged as robust preclinical models; however, their potential to predict clinical outcomes in patients has remained unclear. We report on a living biobank of PDOs from metastatic, heavily pretreated colorectal and gastroesophageal cancer patients recruited in phase 1/2 clinical trials. Phenotypic and genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. Molecular profiling of tumor organoids was matched to drug-screening results, suggesting that PDOs could complement existing approaches in defining cancer vulnerabilities and improving treatment responses. We compared responses to anticancer agents ex vivo in organoids and PDO-based orthotopic mouse tumor xenograft models with the responses of the patients in clinical trials. Our data suggest that PDOs can recapitulate patient responses in the clinic and could be implemented in personalized medicine programs.

Patient-derived organoids model treatment response of metastatic gastrointestinal cancers

As high-throughput sequencing platforms produce longer and longer reads, sequences generated from short inserts, such as those obtained from fossil and degraded material, are increasingly expected to contain adapter sequences. Efficient adapter trimming algorithms are also needed to process the growing amount of data generated per sequencing run. We introduce AdapterRemoval v2, a major revision of AdapterRemoval v1, which introduces (i) striking improvements in throughput, through the use of single instruction, multiple data (SIMD; SSE1 and SSE2) instructions and multi-threading support, (ii) the ability to handle datasets containing reads or read-pairs with different adapters or adapter pairs, (iii) simultaneous demultiplexing and adapter trimming, (iv) the ability to reconstruct adapter sequences from paired-end reads for poorly documented data sets, and (v) native gzip and bzip2 support. We show that AdapterRemoval v2 compares favorably with existing tools, while offering superior throughput to most alternatives examined here, both for single and multi-threaded operations.

/pdf/adapterremoval-v2-rapid-adapter-trimming-identification-and-1ml1rcsk5u.pdf

AdapterRemoval v2: rapid adapter trimming, identification, and read merging.

Quality control (QC) and preprocessing are essential steps for sequencing data analysis to ensure the accuracy of results. However, existing tools cannot provide a satisfying solution with integrated comprehensive functions, proper architectures, and highly scalable acceleration. In this article, we demonstrate SOAPnuke as a tool with abundant functions for a "QC-Preprocess-QC" workflow and MapReduce acceleration framework. Four modules with different preprocessing functions are designed for processing datasets from genomic, small RNA, Digital Gene Expression, and metagenomic experiments, respectively. As a workflow-like tool, SOAPnuke centralizes processing functions into 1 executable and predefines their order to avoid the necessity of reformatting different files when switching tools. Furthermore, the MapReduce framework enables large scalability to distribute all the processing works to an entire compute cluster.We conducted a benchmarking where SOAPnuke and other tools are used to preprocess a ∼30× NA12878 dataset published by GIAB. The standalone operation of SOAPnuke struck a balance between resource occupancy and performance. When accelerated on 16 working nodes with MapReduce, SOAPnuke achieved ∼5.7 times the fastest speed of other tools.

SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data.

INTRODUCTION Cancer is one of the leading causes of death worldwide. Although the 2% of the human genome that encodes proteins has been extensively studied, much remains to be learned about the noncoding genome and gene regulation in cancer. Genes are turned on and off in the proper cell types and cell states by transcription factor (TF) proteins acting on DNA regulatory elements that are scattered over the vast noncoding genome and exert long-range influences. The Cancer Genome Atlas (TCGA) is a global consortium that aims to accelerate the understanding of the molecular basis of cancer. TCGA has systematically collected DNA mutation, methylation, RNA expression, and other comprehensive datasets from primary human cancer tissue. TCGA has served as an invaluable resource for the identification of genomic aberrations, altered transcriptional networks, and cancer subtypes. Nonetheless, the gene regulatory landscapes of these tumors have largely been inferred through indirect means. RATIONALE A hallmark of active DNA regulatory elements is chromatin accessibility. Eukaryotic genomes are compacted in chromatin, a complex of DNA and proteins, and only the active regulatory elements are accessible by the cell’s machinery such as TFs. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) quantifies DNA accessibility through the use of transposase enzymes that insert sequencing adapters at these accessible chromatin sites. ATAC-seq enables the genome-wide profiling of TF binding events that orchestrate gene expression programs and give a cell its identity. RESULTS We generated high-quality ATAC-seq data in 410 tumor samples from TCGA, identifying diverse regulatory landscapes across 23 cancer types. These chromatin accessibility profiles identify cancer- and tissue-specific DNA regulatory elements that enable classification of tumor subtypes with newly recognized prognostic importance. We identify distinct TF activities in cancer based on differences in the inferred patterns of TF-DNA interaction and gene expression. Genome-wide correlation of gene expression and chromatin accessibility predicts tens of thousands of putative interactions between distal regulatory elements and gene promoters, including key oncogenes and targets in cancer immunotherapy, such as MYC , SRC , BCL2 , and PDL1 . Moreover, these regulatory interactions inform known genetic risk loci linked to cancer predisposition, nominating biochemical mechanisms and target genes for many cancer-linked genetic variants. Lastly, integration with mutation profiling by whole-genome sequencing identifies cancer-relevant noncoding mutations that are associated with altered gene expression. A single-base mutation located 12 kilobases upstream of the FGD4 gene, a regulator of the actin cytoskeleton, generates a putative de novo binding site for an NKX TF and is associated with an increase in chromatin accessibility and a concomitant increase in FGD4 gene expression. CONCLUSION The accessible genome of primary human cancers provides a wealth of information on the susceptibility, mechanisms, prognosis, and potential therapeutic strategies of diverse cancer types. Prediction of interactions between DNA regulatory elements and gene promoters sets the stage for future integrative gene regulatory network analyses. The discovery of hundreds of noncoding somatic mutations that exhibit allele-specific regulatory effects suggests a pervasive mechanism for cancer cells to manipulate gene expression and increase cellular fitness. These data may serve as a foundational resource for the cancer research community.

The chromatin accessibility landscape of primary human cancers

The quantity of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is a robust prognostic factor for improved patient survival, particularly in triple-negative and HER2-overexpressing BC subtypes1. Although T cells are the predominant TIL population2, the relationship between quantitative and qualitative differences in T cell subpopulations and patient prognosis remains unknown. We performed single-cell RNA sequencing (scRNA-seq) of 6,311 T cells isolated from human BCs and show that significant heterogeneity exists in the infiltrating T cell population. We demonstrate that BCs with a high number of TILs contained CD8+ T cells with features of tissue-resident memory T (TRM) cell differentiation and that these CD8+ TRM cells expressed high levels of immune checkpoint molecules and effector proteins. A CD8+ TRM gene signature developed from the scRNA-seq data was significantly associated with improved patient survival in early-stage triple-negative breast cancer (TNBC) and provided better prognostication than CD8 expression alone. Our data suggest that CD8+ TRM cells contribute to BC immunosurveillance and are the key targets of modulation by immune checkpoint inhibition. Further understanding of the development, maintenance and regulation of TRM cells will be crucial for successful immunotherapeutic development in BC.

Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis

Adapter trimming is a prerequisite step for analyzing next-generation sequencing (NGS) data when the reads are longer than the target DNA/RNA fragments. Although typically used in small RNA sequencing, adapter trimming is also used widely in other applications, such as genome DNA sequencing and transcriptome RNA/cDNA sequencing, where fragments shorter than a read are sometimes obtained because of the limitations of NGS protocols. For the newly emerged Nextera long mate-pair (LMP) protocol, junction adapters are located in the middle of all properly constructed fragments; hence, adapter trimming is essential to gain the correct paired reads. However, our investigations have shown that few adapter trimming tools meet both efficiency and accuracy requirements simultaneously. The performances of these tools can be even worse for paired-end and/or mate-pair sequencing. To improve the efficiency of adapter trimming, we devised a novel algorithm, the bit-masked k-difference matching algorithm, which has O(k n) expected time with O(m) space, where k is the maximum number of differences allowed, n is the read length, and m is the adapter length. This algorithm makes it possible to fully enumerate all candidates that meet a specified threshold, e.g. error ratio, within a short period of time. To improve the accuracy of this algorithm, we designed a simple and easy-to-explain statistical scoring scheme to evaluate candidates in the pattern matching step. We also devised scoring schemes to fully exploit the paired-end/mate-pair information when it is applicable. All these features have been implemented in an industry-standard tool named Skewer (
 https://sourceforge.net/projects/skewer
 ). Experiments on simulated data, real data of small RNA sequencing, paired-end RNA sequencing, and Nextera LMP sequencing showed that Skewer outperforms all other similar tools that have the same utility. Further, Skewer is considerably faster than other tools that have comparative accuracies; namely, one times faster for single-end sequencing, more than 12 times faster for paired-end sequencing, and 49% faster for LMP sequencing. Skewer achieved as yet unmatched accuracies for adapter trimming with low time bound.

https://link.springer.com/content/pdf/10.1186%2F1471-2105-15-182.pdf

Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads.

Bactrocera dorsalis is an invasive polyphagous pest causing considerable ecological and economic damage worldwide. We report a high-quality chromosome-level genome assembly and combine various transcriptome data to explore the molecular mechanisms of its rapid adaptation to new environments. The expansions of the DDE transposase superfamily and key gene families related to environmental adaptation and enrichment of the expanded and unique gene families in metabolism and defence response pathways explain its environmental adaptability. The relatively high but not significantly different expression of heat-shock proteins, regardless of the environmental conditions, suggests an intrinsic mechanism underlying its adaptation to high temperatures. The mitogen-activated protein kinase pathway plays a key role in adaptation to new environments. The prevalence of duplicated genes in its genome explains the diversity in the B. dorsalis complex. These findings provide insights into the genetic basis of the invasiveness and diversity of B. dorsalis, explaining its rapid adaptation and expansion. 

/pdf/chromosome-level-genome-assembly-of-bactrocera-dorsalis-2ep1sanv.pdf

Chromosome-level genome assembly of Bactrocera dorsalis reveals its adaptation and invasion mechanisms

The use of omics techniques to analyze the differences between genetic engineering organisms and their parents can identify unintended effects and explore whether such unintended effects will have negative consequences. In order to evaluate whether genetic engineering will cause changes in crops beyond the changes introduced by conventional plant breeding, we compared the extent of transcriptome and metabolome modification in the leaves of three lines developed by RNA interference (RNAi)-based genetic engineering and three lines developed by conventional breeding. The results showed that both types of plant breeding methods can manifest changes at the short interfering RNA (siRNA), transcriptomic, and metabolic levels. Relative expression analysis of potential off-target gene revealed that there was no broad gene decline in the three RNAi-based genetic engineering lines. We found that the number of DEGs and DAMs between RNAi-based genetic engineering lines and the parental line was less than that between conventional breeding lines. These unique DEGs and DAMs between RNAi-based genetic engineering lines and the parental lines were not enriched in detrimental metabolic pathways. The results suggest that RNAi-based genetic engineering do not cause unintended effects beyond those found in conventional breeding in maize.

RNA Interference-Based Genetic Engineering Maize Resistant to Apolygus lucorum Does Not Manifest Unpredictable Unintended Effects Relative to Conventional Breeding: Short Interfering RNA, Transcriptome, and Metabolome Analysis

The protoplast experimental system eis an effective tool for functional genomics and cell fusion breeding. However, the physiological and molecular mechanisms of protoplast response to enzymolysis are not clear, which has become a major obstacle to protoplast regeneration. Here, we used physiological, cytological, proteomics and gene expression analysis to compare the young leaves of sugarcane and enzymolized protoplasts. After enzymatic digestion, we obtained protoplasts with viability of > 90%. Meanwhile, the content of malondialdehyde, an oxidation product, increased in the protoplasts following enzymolysis, and the activity of antioxidant enzymes, such as peroxidase (POD), catalase (CAT), acid peroxidase (APX), and O2-, significantly decreased. Cytologic analysis results showed that, post enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed several short rod-like structures in protoplasts. In this study, a proteomics approaches was used to identify proteins of protoplasts in response to the enzymatic digestion process. GO, KEGG, and KOG enrichment analyses revealed that the abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which allow for protein biosynthesis or degradation. RT-qPCR analysis revealed that the expression of osmotic stress resistance genes, such as DREB, WRKY, MAPK4, and NAC, was upregulated, while that of key regeneration genes, such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA, and GAUT, was significantly downregulated in the protoplasts. Hierarchical clustering and identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. Our findings can facilitate the development of a standard system to produce regenerated protoplasts using molecular markers and antibody detection of enzymolysis.

/pdf/an-integrated-physiology-cytology-and-proteomics-analysis-3c0l2qf6.pdf

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

The protoplast experimental system has been becoming a powerful tool for functional genomics and cell fusion breeding. However, the physiology and molecular mechanism during enzymolysis is not completely understood and has become a major obstacle to protoplast regeneration. Our study used physiological, cytology, iTRAQ (Isobaric Tags for Relative and Absolute Quantification) -based proteomic and RT-PCR analyses to compare the young leaves of sugarcane (ROC22) and protoplasts of more than 90% viability. We found that oxidation product MDA content increased in the protoplasts after enzymolysis and several antioxidant enzymes such as POD, CAT, APX, and O2- content significantly decreased. The cytology results showed that after enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed many short rod-like structures in protoplasts. The proteomic results showed that 1,477 differential proteins were down-regulated and 810 were up-regulated after enzymolysis of sugarcane young leaves. The GO terms, KEGG and KOG enrichment analysis revealed that differentially abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which would allow protein biosynthesis or / degradation. The RT-PCR analysis revealed the expression of osmotic stress resistance genes such as DREB, WRKY, MAPK4, and NAC were up-regulated. Meanwhile, the expression of key regeneration genes such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA and GAUT were significantly down-regulated in the protoplasts. Hierarchical clustering, identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. We used a variety of methods to figure out how young sugarcane leaves react to enzymes.

Shuifang Zhu

Papers

Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads.

Chromosome-level genome assembly of Bactrocera dorsalis reveals its adaptation and invasion mechanisms

RNA Interference-Based Genetic Engineering Maize Resistant to Apolygus lucorum Does Not Manifest Unpredictable Unintended Effects Relative to Conventional Breeding: Short Interfering RNA, Transcriptome, and Metabolome Analysis

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

An Integrated Physiological, Cytology and Proteomics Reveals Network of Sugarcane Protoplasts Responses to Enzymolysis