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Background Approximately 2-7% of patients with non-small cell lung cancer harbor anaplastic lymphoma kinase (ALK) rearrangement events. Of note, typical ALK actionable rearrangements are sensitive to treatment with tyrosine kinase inhibitors (TKIs). However, different types of ALK fusion influence the clinical outcomes of this therapeutic approach. Approximately 10-40% of patients with ALK-fusion positive non-small cell lung cancer do not response to ALK-TKI therapy. Therefore, it is important to accurately identify the types of ALK rearrangement for appropriate selection of clinical treatment. Case report Using a DNA-targeted next-generation sequencing technique, we found a novel solute carrier family 8 member A1 (SLC8A1)-ALK fusion type in a patient with lung adenocarcinoma. Further reverse transcriptase-polymerase chain reaction and Sanger sequencing demonstrated the rearrangement as a B-cell CLL/lymphoma 11A (BCL11A)-ALK fusion at the transcriptional level. The patient showed a rapid and strong response to treatment with crizotinib, which lasted for 9 months. The patient also responded well to treatment with alectinib after developed resistance to crizotinib. Conclusion A strategy combining DNA-targeted next-generation sequencing with RNA reverse transcriptase-polymerase chain reaction and sequencing, besides fluorescence in situ hybridization and immunohistochemistry, may provide an effective and practical solution for correct identification of partner genes and fusion structures in the diagnosis of ALK rearrangements, particularly for non-canonical expression patterns of ALK fusion events. The combined approach may lead to more benefits for patients.

Small-cell lung cancer (SCLC) is an exceptionally lethal malignancy characterized by extremely high alteration rates and tumor heterogeneity, which limits therapeutic options. In contrast to non-small-cell lung cancer that develops rapidly with precision oncology, SCLC still remains outside the realm of precision medicine. No recurrent and actionable mutations have been detected. Additionally, a paucity of substantive tumor specimens has made it even more difficult to classify SCLC subtypes based on genetic background. We therefore carried out whole-exome sequencing (WES) on the largest available Chinese SCLC cohort. For the first time, we partitioned SCLC patients into three clusters with different genomic alteration profiles and clinical features based on their mutational signatures. We showed that these clusters presented differences in intratumor heterogeneity and genome instability. Moreover, a wide existence of mutually exclusive gene alterations, typically within similar biological functions, was detected and suggested a high SCLC intertumoral heterogeneity. Particularly, Cluster 1 presented the greatest potential to benefit from immunotherapy, and Cluster 3 constituted recalcitrant SCLC, warranting biomarker-directed drug development and targeted therapies in clinical trials. Our study would provide an in-depth insight into the genome characteristics of the Chinese SCLC cohort, defining distinct molecular subtypes as well as subtype-specific therapies and biomarkers. We propose tailoring differentiated therapies for distinct molecular subgroups, centering on a personalized precision chemotherapy strategy combined with immunization or targeted therapy for patients with SCLC.

Genome‐wide 5hmC profiling in cell‐free DNA acquired from CRC patients, adenoma patients, and healthy individuals revealed that the differential 5hmC‐modified regions were gathered into four clusters with no overlap, although there are a few overlapped genes shared between the different clusters. CRC patients with adenoma history showed exclusive 5hmC‐gain characteristics, which was consistent with the ‘parallel’ evolution hypothesis in adenoma., Approximately 85% colorectal cancers (CRCs) are thought to evolve through the adenoma‐to‐carcinoma sequence associated with specific molecular alterations, including the 5‐hydroxymethylcytosine (5hmC) signature in circulating cell‐free DNA (cfDNA). To explore colorectal disease progression and evaluate the use of cfDNA as a potential diagnostic factor for CRC screening, here, we performed genome‐wide 5hmC profiling in plasma cfDNA and tissue genomic DNA (gDNA) acquired from 101 samples (63 plasma and 38 tissues), collected from 21 early‐stage CRC patients, 21 AD patients, and 21 healthy controls (HC). The gDNA and cfDNA 5hmC signatures identified in gene bodies and promoter regions in CRC and AD groups were compared with those in HC group. All the differential 5hmC‐modified regions (DhMRs) were gathered into four clusters: Disease‐enriched, AD‐enriched, Disease‐lost, and AD‐lost, with no overlap. AD‐related clusters, AD‐enriched and AD‐lost, displayed the unique 5hmC signals in AD patients. Disease‐enriched and Disease‐lost clusters indicated the general 5hmC changes when colorectal lesions occurred. Cancer patients with a confirmable adenoma history segmentally gathered in AD‐enriched clusters. KEGG functional enrichment and GO analyses determined distinct differential 5hmC‐modified profiles in cfDNA of HC individuals, AD, and CRC patients. All patients had comprehensive 5hmC signatures where Disease‐enriched and Disease‐lost DhMR clusters demonstrated similar epigenetic modifications, while AD‐enriched and AD‐lost DhMR clusters indicated complicated subpopulations in adenoma. Analysis of CRC patients with adenoma history showed exclusive 5hmC‐gain characteristics, consistent with the ‘parallel’ evolution hypothesis in adenoma, either developed through the adenoma‐to‐carcinoma sequence or not. These findings deepen our understanding of colorectal disease and suggest that the 5hmC modifications of different pathological subtypes (cancer patients with or without adenoma history) could be used to screen early‐stage CRC and assess adenoma malignancy with large‐scale follow‐up studies in the future.

Background Esophageal cancer is one of globally high incidence and mortality disease. Its early stage has rarely obvious symptoms. Compared to conventional endoscopy diagnosis, liquid biopsy is an emerging non-invasive method for cancer early detection. Methods We enrolled 100 esophageal squamous cell carcinoma (ESCC) patients and 71 healthy individuals as a Southern China cohort and performed 5-hydroxymethylcytosine (5hmC) sequencing on their plasma cell-free DNA (cfDNA). A Northern cohort of cfDNA 5hmC dataset with 150 ESCC patients and 183 health individuals were downloaded for validation. A diagnostic model was firstly developed based on cfDNA 5hmC signatures and then improved by low-pass whole genome sequencing (WGS) features of cfDNA. Results Conserved cfDNA 5hmC modification motifs were observed in the two independent ESCC cohorts. A diagnostic model with 273 5hmC features based on randomly-selected two-thirds samples of the Southern China cohort was validated independently in the left one-thirds samples and the whole Northern China cohort, achieved an AUC of 0.810 and 0.862 with sensitivities of 69.3–74.3% and specificities of 82.4–90.7%, respectively. The performance was well maintained in Stage I to Stage IV, with accuracy of 70%-100%, but low in Stage 0, with accuracy of 33.3%. Low-pass WGS of cfDNA improved the AUC to 0.934 with a sensitivity of 82.4%, a specificity of 88.2% and an accuracy of 84.3%, particularly significantly in Stage 0 with the accuracy up to 80%. Conclusions This study suggests that the blood-based 5hmC integrated with low-pass WGS model could improve the accurate diagnosis of early stage ESCC, particularly for very early stage ESCC.

Stage I non-small cell lung cancer (NSCLC) presents diverse outcomes. To identify molecular features leading to tumor recurrence in early-stage NSCLC, we perform multiregional whole-exome sequencing (WES), RNA sequencing, and plasma-targeted circulating tumor DNA (ctDNA) detection analysis between recurrent and recurrent-free stage I NSCLC patients (CHN-P cohort) who had undergone R0 resection with a median 5-year follow-up time. Integrated analysis indicates that the multidimensional clinical and genomic model can stratify the prognosis of stage I NSCLC in both CHN-P and EUR-T cohorts and correlates with positive pre-surgical deep next generation sequencing (NGS) ctDNA detection. Increased genomic instability related to DNA interstrand crosslinks and double-strand break repair processes is significantly associated with early tumor relapse. This study reveals important molecular insights into stage I NSCLC and may inform clinical postoperative treatment and follow-up strategies.

Influenza is a persistent threat to human health and there is a continuing requirement for updating anti-influenza strategies. Initiated by observations of different endoplasmic reticulum (ER) responses of host to seasonal H1N1 and highly pathogenic avian influenza (HPAI) A H5N1 infections, we identified an alternative antiviral role of tauroursodeoxycholic acid (TUDCA), a clinically available ER stress inhibitor, both in vitro and in vivo. Rather than modulating ER stress in host cells, TUDCA abolished the proton conductivity of viral M2 by disrupting its oligomeric states, which induces inefficient viral infection. We also showed that M2 penetrated cells, whose intracellular uptake depended on its proton channel activity, an effect observed in both TUDCA and M2 inhibitor amantadine. The identification and application of TUDCA as an inhibitor of M2 proton channel will expand our understanding of IAV biology and complement current anti-IAV arsenals.

The red algae Kappaphycus alvarezii is the most important aquaculture species in Kappaphycus, widely distributed in tropical waters, and it has become the main crop of carrageenan production at present. The mechanisms of adaptation for high temperature, high salinity environments and carbohydrate metabolism may provide an important inspiration for marine algae study. Scientific background knowledge such as genomic data will be also essential to improve disease resistance and production traits of K. alvarezii. 43.28 Gb short paired-end reads and 18.52 Gb single-molecule long reads of K. alvarezii were generated by Illumina HiSeq platform and Pacbio RSII platform respectively. The de novo genome assembly was performed using Falcon_unzip and Canu software, and then improved with Pilon. The final assembled genome (336 Mb) consists of 888 scaffolds with a contig N50 of 849 Kb. Further annotation analyses predicted 21,422 protein-coding genes, with 61.28% functionally annotated. Here we report the draft genome and annotations of K. alvarezii, which are valuable resources for future genomic and genetic studies in Kappaphycus and other algae.

A portion of stage I non-small cell lung cancer (NSCLC) patients who have received R0 resection experience unexpected tumor recurrence during follow up. Some of them even recurred within 12 months after surgery, which is unable to predict by any clinical characteristics. Few study investigates the molecular features of such short-term recurrent tumors. Multiomics analysis were performed on 81 stage I NSCLC patients who had received R0 resection, based on deep whole-exome sequencing (average depth >500x) on multi-region 239 tumor tissue samples and 81 matched-adjacent normal specimens, as well as RNA sequencing and plasma targeted circulating tumor DNA detection. Dynamic dimension comparison of genomic alteration features between tumor recurrence and non-recurrence patients was made according to 60-months median time of follow up. Published WES datasets of Stage I NSCLCs of 131 East Asian (EAS LUADs), 62 European (TRACERx) and 277 American patients (TCGA) were extracted as validation. 60.5% patients were non-smokers. Lower chromosomal instability was found in our cohort characterized by less trunk CNVs and lower CNV coverage on genome as compared to the European cohort. Age-related signature mutations significantly accumulated in recurrence-free tumors. Somatic mutations occurred in DNA damage repair pathways were more likely to cause tumor recurrence (p=0.04), particularly in homologous recombination (HRR) (p=0.017) and translesion synthesis (TLS) (p=0.009) pathways. Dynamic analysis discovered the essential genes in Fanconi anemia (FA), TLS and HRR pathways that coordinate together for DNA interstrand crosslink (ICL) recognition and faithful repair of DNA double strand breaks (DSBs) during DNA replication stress frequently mutated in early tumor recurrence patients (n=7/11, 63%). Twenty-two focal CNV-driver genes, including cell proliferation-related RECQL4 (8q24.3), PIK3CA (3q26.32), BCL9 (1q21.2), TBL1XR1 (3q26.32) frequently amplified in recurrent tumors, as well as BCL3 and CBLC (19q13.31-32) in the early recurrent tumors. RNA-seq data demonstrated cell cycle pathway significantly up-regulated in recurrent tumors. More TMB, CNV fraction and genome doubling events in FA-TLS-HRR alteration patients verified genomic instability caused by dysfunction of cell cycle control and ICL-DSB repair contributing to early tumor recurrence. Integrative model cross clinical and molecular features stratified stage I patient prognosis in both our cohort and European cohort, in which and high risk patients were more detected by circulating tumor DNA. This is the first study to dynamically investigate the genomic features specifically in stage I lung NSCLC according to the precise tumor recurrence time, which provides important clues to postoperative treatment strategy and drug target research. Citation Format: Kezhong Chen, Airong Yang, Shuangxiu Wu, Yuntao Nie, Haifeng Shen, Jian Bai, Lin Wu, Fan Yang, Jun Wang. Spatio-temporal multiomics analysis reveals distinct molecular features in recurrent stage I non-small cell lung cancers after R0 tumor resection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 618.

To improve H2 production, the green algae Chlamydomonas reinhardtii cc849 was co-cultured with Azotobacter chroococcum. The maximum H2 production of the co-culture was 350% greater than that of the pure algal cultures under optimal H2 production conditions. The maximum growth and the respiratory rate of the co-cultures were about 320 and 300% of the controls, and the dissolved O2 of co-cultures was decreased 74%. Furthermore, the in vitro maximum hydrogenase activity of the co-culture was 250% greater than that of the control, and the in vivo maximum hydrogenase activity of the co-culture was 1.4-fold greater than that of the control. In addition, the maximum starch content of co-culture was 1400% that of the control. Azotobacter chroococcum improved the H2 production of the co-cultures by decreasing the O2 content and increasing the growth and starch content of the algae and the hydrogenase activity of the co-cultures relative to those of pure algal cultures.

The evolutionary dynamics of tumor‐associated neoantigens carry information about drug sensitivity and resistance to the immune checkpoint blockade (ICB). However, the spectrum of somatic mutations is highly heterogeneous among patients, making it difficult to track neoantigens by circulating tumor DNA (ctDNA) sequencing using “one size fits all” commercial gene panels. Thus, individually customized panels (ICPs) are needed to track neoantigen evolution comprehensively during ICB treatment. Dominant neoantigens are predicted from whole exome sequencing data for treatment‐naïve tumor tissues. Panels targeting predicted neoantigens are used for personalized ctDNA sequencing. Analyzing ten patients with non‐small cell lung cancer, ICPs are effective for tracking most predicted dominant neoantigens (80–100%) in serial peripheral blood samples, and to detect substantially more genes (18–30) than the capacity of current commercial gene panels. A more than 50% decrease in ctDNA concentration after eight weeks of ICB administration is associated with favorable progression‐free survival. Furthermore, at the individual level, the magnitude of the early ctDNA response is correlated with the subsequent change in tumor burden. The application of ICP‐based ctDNA sequencing is expected to improve the understanding of ICB‐driven tumor evolution and to provide personalized management strategies that optimize the clinical benefits of immunotherapies., Inter‐individual heterogeneity of mutation profiling is the primary obstacle in effective in vivo monitoring of mutation‐directed T‐cell killing using ctDNA sequencing. Herein, a customized sequencing strategy is developed that mainly targets predicted neoantigen‐coding mutations. This approach broadens the ctDNA sequencing coverage and is expected to improve personalized management strategies that optimize the clinical benefits from checkpoint immunotherapies.

BackgroundThe 1000 Plant transcriptomes initiative (1KP) explored genetic diversity by sequencing RNA from 1,342 samples representing 1,173 species of green plants (Viridiplantae).FindingsThis data release accompanies the initiative's final/capstone publication on a set of 3 analyses inferring species trees, whole genome duplications, and gene family expansions. These and previous analyses are based on de novo transcriptome assemblies and related gene predictions. Here, we assess their data and assembly qualities and explain how we detected potential contaminations.ConclusionsThese data will be useful to plant and/or evolutionary scientists with interests in particular gene families, either across the green plant tree of life or in more focused lineages.

The 1000 Plants (1KP) initiative explored the genetic diversity of green plants (Viridiplantae) by sequencing RNA from 1,342 samples representing 1,173 species. All of the analyses done for the 1KP capstone, and previous studies on subsets of these data, are based on a series of de novo transcriptome assemblies and related outputs that will be described in this publication. We expect that these data will also be useful to other researchers with interests in specific gene families, either across the green plant tree of life or in more focused lineages. These protocols provide additional details on the sample and sequence processing, hopefully providing insight for reproducibility and tools for other researchers working on plant transcriptomics and evolution.

SOAP-denovo-trans assembly protocol used to assemble plant transcriptomes for the 1KP project.

Illumina GAII Library Construction and Sequencing for RNA Seq

Hiseq 2000 Library Construction and Sequencing for RNA Seq

Background Panax ginseng C. A. Mey is one of famous medicinal herb plant species. Its major bioactive compounds are various ginsenosides in roots and rhizomes. It is commonly accepted that ginsenosides are synthesized from terpene precursors, IPP and DMAPP, through the cytoplasmic mevalonate (MVA) pathway. Another plastic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was proved also contributing to ginsenoside generation in the roots of P. ginseng by using specific chemical inhibitors recently. But their gene expression characteristics are still under reveal in P. ginseng. With the development of the high-throughput next generation sequencing (NGS) technologies, we have opportunities to discover more about the complex ginsenoside biosynthesis pathways in P. ginseng. Results We carried out deep RNA sequencing and comprehensive analyses on the ginseng root samples of 1–5 years old and five different tissues of 5 years old ginseng plants. The de novo assembly totally generated 48,165 unigenes, including 380 genes related to ginsenoside biosynthesis and all the genes encoding the enzymes of the MEP pathway and the MVA pathway. We further illustrated the gene expression profiles related to ginsenoside biosynthesis among 1–5 year-old roots and different tissues of 5 year-old ginseng plants. Particularly for the first time, we revealed that the gene transcript abundances of the MEP pathway were similar to those of the MVA pathway in ginseng roots but higher in ginseng leaves. The IspD was predicated to be the rate-limiting enzyme in the MEP pathway through both co-expression network and gene expression profile analyses. Conclusions At the transcriptional level, the MEP pathway has similar contribution to ginsenoside biosynthesis in ginseng roots, but much higher in ginseng leaves, compared with the MVA pathway. The IspD might be the key enzyme for ginsenoside generation through the MEP pathway. These results provide new information for further synthetic biology study on ginsenoside metabolic regulation. Electronic supplementary material The online version of this article (10.1186/s12864-019-5718-x) contains supplementary material, which is available to authorized users.

Figure S1. Photographs of P. ginseng plants collected in the field and the dissected ginseng tissues used in this study (including main root barks, main root cores, lateral roots, root hairs, rhizomes, stems and leaves). A, the photograph of P. ginseng root. B, dissections of a ginseng root to show the tissues of main roots, small roots (diameter between 2 mm and 3.5 mm), lateral roots (diameter between 1 mm and 2 mm), root hairs (diameter less than 1 mm) and rhizomes. C, dissections of a ginseng root to show the tissues of the main root bark and main root cores. D. 1 year to 5 years old ginseng plants collected in the field. The scale bar was indicated in each pattern. (PDF 130 kb)

Table S1. Statistics of quality control on RNA-seq data of 1â 5â years old root samples and other tissue samples of 5â years old of P. ginseng. (PDF 68 kb)

Table S3. Comparison of ginseng CDS gene sets between published genomic dataset and our assembled RNA-seq dataset of P. ginseng (PDF 66â kb) (PDF 66 kb)

Table S4. Comparison analyses on the gene numbers annotated in ginsenoside biosynthesis-related pathways between our assembled RNA-seqdataset and other published datasets. (PDF 50 kb)

Table S5. Statistics of the isoform numbers of the genes related to ginsenoside biosynthesis among different Panax species and W. somnifera based on RNA-seq data. (PDF 90 kb)

Figure S2. Saturation curve of transcriptome sequencing data of each samples of P. ginseng using BWA software. R11 and R12, two biological duplication samples of 1 year-old root samples. R21 and R22, two biological duplication samples of 2 year-old root samples. R3a and R35, two biological duplication samples of 3 year-old root samples. R41 and R42, two biological duplication samples of 4 year-old root samples. R52 and R5b, two biological duplication samples of 5 year-old root samples. Rc, root cores. Rt, lateral roots. Bs, rhizomes. St, stems. Lf, leaves. (PDF 125 kb)

Table S2. Primer sequences of the genes of the MEP pathway of P. ginseng for qPCR verification (PDF 57 kb)

Figure S3. Histograms of gene transcription abundance distribution of RNA-seq samples of P. ginseng using RSEM software. R11 and R12, two biological duplication samples of 1 year-old root samples. R21 and R22, two biological duplication samples of 2 year-old root samples. R3a and R35, two biological duplication samples of 3 year-old root samples. R41 and R42, two biological duplication samples of 4 year-old root samples. R52 and R5b, two biological duplication samples of 5 year-old root samples. Rc, root cores. Rt, lateral roots. Bs, rhizomes. St, stems. Lf, leaves. (PDF 66 kb)

e21021 Background: Circulating tumour cells (CTCs) have important applications in clinical practice on early tumour diagnosis, prognostic prediction and treatment evaluation. Platinum-based chemotherapy is the first-line treatment for non-small cell lung cancer (NSCLC) patients without targeted drugs. But most patients progressed after a period of treatment. Therefore, to dissect the genetic information contributing to drug resistance and tumour progression in CTCs is valuable for guidance of treatment adjustment. Methods: In this study, we enrolled 9 NSCLC patients treated with platinum-based chemotherapy. For each patient, 10 CTCs were isolated after progression to perform a single cell-level whole-exome sequencing (WES). Meanwhile the patients’ paired primary-diagnosed FFPE sample and progressive biopsy specimens were also performed WES. Results: Comparisons of WES data between primary and progressive specimens, as well as CTCs revealed that more than half patients’ tumour mutation burden (TMB) increased after progression. Dozes to hundreds of single-nucleotide variants (SNVs) and insertions or deletions (Indels) were detected in the CTCs. Slightly more proportion of SNVs/Indels in CTCs shared with paired primary tumours (1.2%-23.1%) than with progressive samples (0.6%-11.7%). Conclusions: Functional annotation on SNVs/Indels showed that CTCs not only harboured cancer driver gene mutations, such as EGFR and TP53, shared with primary and/or progressive tumours, but also harboured chemotherapy-resistance and stem cell-related gene mutations, including SHKBP1, NUMA1, ZNF143, MUC16, ORC1, PON1, PELP1, etc. which have crucial roles in drug resistance and poor prognosis for NSCLCs. Thus, detection of genetic information in CTCs was necessary for guidance of individual therapy and drug resistance study.

Three algae strains—Chlamydomonas reinhardtii hemHc-lbac (transgenic), cc124 and cc503—were co-cultured with Bradyrhizobium japonicum to improve H2 production. The maximum H2 productions of the three different co-cultures were 3.5-fold, 17-fold and 4.4-fold of the pure algal cultures. Meanwhile, the biomass of each of the algae strains was increased by B. japonicum. Furthermore, the respiratory rates of the co-cultures were 1.4-fold, 1.2-fold and 2.6-fold higher than the controls, respectively; the in vitro maximum hydrogenase activities of the co-cultures were 1.1-fold higher, equal to 2.4-fold higher than those of the controls, respectively, and the in vivo maximum hydrogenase activities of the co-cultures were 1.5-fold, 3.8-fold and 2.1-fold higher than the controls, respectively. The maximum starch content of the co-cultures were 8.3-fold, 8.4-fold and 4.4-fold higher than the content of the controls, respectively. Therefore, B. japonicum improved the H2 production of the co-cultures by increasing the biomass, respiratory rates, hydrogen activity and starch content of the co-cultures relative to those of pure algae cultures.

Colorectal cancer (CRC) is a serious malignancy with the third incidence and second mortality throughout the world. 5-Hydroxymethylcytosine (5hmC) signatures in circulating cell-free DNA (cfDNA) is an important epigenetic mark linked with human cancer pathogenesis. Here we explored the genome-wide 5-hydroxymethylcytosine profiling in the plasma cfDNA and tissue genomic DNA (gDNA) acquired from 101 samples (63 from plasma and 38 from tissues), collected from 21 CRC patients, 21 precancerous adenoma (AD) patients and 21 healthy control individuals (HC). 5hmC modifications exhibited distinct features in promoter and gene body regions in all 5 groups (3 groups from plasma and 2 groups from tissues), which offered a global overview of epigenetic changes of colon pathogenic statuses between early stage CRC and AD. Adenoma showed an independent property in the 5hmC profiles compared to cancer and healthy control. The 5hmC signatures in tumor were more comprehensive and could be divided into two clusters, one was similar to the healthy individuals and the other was similar to the AD patients. The results indicated that the candidate biomarkers to distinguish colorectal cancer from the blood-test population should be generated in two ways, one focused on the differential 5hmC features between CRC patients and healthy individuals; the other should detect the adenoma and tumor patients more extensive according to their precise pathological diagnosis and clinical information. Candidate cfDNA 5hmC biomarkers were further identified in the differential 5hmC modification regions between CRC and HC with a higher sensitivity than the classical CEA marker in non-invasive blood-based diagnosis. We suggested that the differentially enriched 5hmC regions in cfDNA were potential biomarkers to track colorectal cancer, even precancerous adenoma, with further large-scale follow-up studies. Citation Format: Zewen Xiao, Jiayan Wu, Chunlong Wu, Man Li, Fuming Sun, Lu Zheng, Gaojing Liu, Xiaoling Li, Zhiyuan Yun, Jiebing Tang, Yang Yu, Shengnan Luo, Wenji Sun, Xiaohong Feng, Qian Cheng, Xue Tao, Jian Bai, Lin Wu, Shuangxiu Wu, Ji Tao. 5-Hydroxymethylcytosine signatures in cell-free DNA as a potential biomarker for colorectal cancer andprecancerous adenoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4597.

The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SPn EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots.

The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.

The transgenic alga lba of Chlamydomonas reinhardtii yielded H2 with 50%–180% higher than the control strain. Further experiments showed that photosynthetic rates and photosynthetic reaction center II's photochemical capacities of the transgenic algae obviously decreased 33.4%–85.9% and 30.0%–51.7%, respectively, compared with those of the control. On the contrary, respiration rates of the transgenic algae significantly increased, with 40.0%–200.0% higher than those of the control. Furthermore, starch contents of the transgenic algae were also improved significantly by 79.1%–592.8% compared with the control. Therefore, the reason of H2 yield improvement of the transgenic alga lba is not only due to its decrease of photosynthetic capacity and increase of the respiration rate, but also due to the metabolic changes related to starch metabolism, photosynthesis and respiration which is possibly caused by hetero-expression of lba gene in chloroplasts of C. reinhardtii, indicating the potential of utilization of lba gene to improve hydrogen yield of micro-green algae.

Endogenous viral elements (EVEs) are host-genomic fragments originated from viral genomes. They have been found universally in animal and plant genomes. Here we carried out a systematic screening and analysis of EVEs in algal genomes and found that EVEs commonly exist in algal genomes. We classified the EVE fragments into three categories according to the length of EVE fragments. Due to the probability of sequence similarity by chance, we ignored the potential function of medium-length EVE fragments. However, longlength EVE fragments probably had capability to encode protein domains or even entire proteins, and some short-length EVE fragments had high similarity with host’s siRNA sequences and possibly served functions of small RNAs. Therefore, short and long EVE fragments might provide regulomic and proteomic novelty to the host’s metabolism and adaptation. We also found several EVE fragments shared by more than 3 algal genomes. By phylogenetic analysis of the shared EVEs and their corresponding species, we found that the integration of viral fragments into host genomes was an ancient event, possibly before the divergence of Chlorophytes and Ochrophytes. Our findings show that there is a frequent genetic flow from viruses to algal genomes. Moreover, study on algal EVEs shed light on the virus-host interaction in large timescale and could also help us understand the balance of marine ecosystems.

Tryptophan synthase (TS, EC 4.2.1.20) catalyzes the last two steps of L-tryptophan biosynthesis. In prokaryotes, tryptophan synthase is a multi-enzyme complex, and it consists of α and β subunit which forms an α-ββ-α complex. In fungi and diatoms, TS is a bifunctional enzyme. Because of the limited genomic and transcriptomic data of algae, there are few studies on TS evolution of algae. Here we analyzed the data of the 1000 Plants Project (1KP), and focused on red algae and brown algae. We found out that the TS of Phaeophyceae were fusion genes, which probably originated from the secondary host nucleus, and that the TS of Rhodophyta contained two genes, TSA and TSB, which both display a possible cyanobacterial origin at the time of primary endosymbiosis. In addition, there were two types of TSB genes (TSB1 and TSB2). Through the multiple sequence alignment of TSB proteins, we found several residues conserved in TSB1 but variable in TSB2 which connect with α subunit. The phenomenon may suggest that the TSB2 sequences of Rhodophyta cannot form stable complex with TSA.

Betaphycus gelatinus, Kappaphycus alvarezii and Eucheuma denticulatum of Family Solieriaceae, Order Gigartinales, Class Rhodophyceae are three important carrageenan-producing red algal species, which produce different types of carrageenans, beta (β)-carrageenan, kappa (κ)-carrageenan and iota (ι)-carrageenan. So far the carrageenan biosynthesis pathway is not fully understood and few information is about the Solieriaceae genome and transcriptome sequence. Here, we performed the de novo transcriptome sequencing, assembly, functional annotation and comparative analysis of these three commercial-valuable species using an Illumina short-sequencing platform Hiseq 2000 and bioinformatic software. Furthermore, we compared the different expression of some unigenes involved in some pathways relevant to carrageenan biosynthesis. We finally found 861 different expressed KEGG orthologs which contained a glycolysis/gluconeogenesis pathway (21 orthologs), carbon fixation in photosynthetic organisms (16 orthologs), galactose metabolism (5 orthologs), and fructose and mannose metabolism (9 orthologs) which are parts of the carbohydrate metabolism. We also found 8 different expressed KEGG orthologs for sulfur metabolism which might be importantly related to biosynthesis of different types of carrageenans. The results presented in this study provided valuable resources for functional genomics annotation and investigation of mechanisms underlying the biosynthesis of carrageenan in Family Solieriaceae.

Most phaeophytes (brown algae) and rhodophytes (red algae) dwell exclusively in marine habitats and play important roles in marine ecology and biodiversity. Many of these brown and red algae are also important resources for industries such as food, medicine and materials due to their unique metabolisms and metabolites. However, many fundamental questions surrounding their origins, early diversification, taxonomy, and special metabolisms remain unsolved because of poor molecular bases in brown and red algal study. As part of the 1 000 Plant Project, the marine macroalgal transcriptomes of 19 Phaeophyceae species and 21 Rhodophyta species from China’s coast were sequenced, covering a total of 2 phyla, 3 classes, 11 orders, and 19 families. An average of 2 Gb per sample and a total 87.3 Gb of RNA-seq raw data were generated. Approximately 15 000 to 25 000 unigenes for each brown algal sample and 5 000 to 10 000 unigenes for each red algal sample were annotated and analyzed. The annotation results showed obvious differences in gene expression and genome characteristics between red algae and brown algae; these differences could even be seen between multicellular and unicellular red algae. The results elucidate some fundamental questions about the phylogenetic taxonomy within phaeophytes and rhodophytes, and also reveal many novel metabolic pathways. These pathways include algal CO2 fixation and particular carbohydrate metabolisms, and related gene/gene family characteristics and evolution in brown and red algae. These findings build on known algal genetic information and significantly improve our understanding of algal biology, biodiversity, evolution, and potential utilization of these marine algae.

Saccharina is one of the most important cold-water living marine brown algal genera. In this study we analyzed the transcriptome of S. japonica, which belongs to the 1 000 Plants (OneKP) Project, by using a next-generation high-throughput DNA sequencing technique. About 5.16 GB of raw data were generated, and 65 536 scaffolds with an average length of 454 bp were assembled with SOAP de novo assembly method. In total, 19 040 unigenes were identified by BLAST; 25 734 scaffolds were clustered into 37 Gene ontology functional groups; 6 760 scaffolds were classified into 25 COG categories, as well as 2 665 scaffolds that were assigned to 306 KEGG pathways. Majority of the unigenes exhibited more similarities to algae including brown algae and diatom than other cyanobacteria, marine diatom, and plant. Saccharina japonica has the outstanding capability to accumulate halogen such as Br and I via halogenation processes from seawater. We acquired 42 different vanadium-dependent haloperoxidases (vHPO) in S. japonica transcriptome data, including 5 segments of vanadium-dependent iodoperoxidase (vIPO) and 37 segments of vanadium-dependent bromoperoxidase (vBPO). Complicated analyses of identified fulllength S. japonica vBPO1 and S. japonica vBPO2 revealed the importance of vBPO among species of brown algae and the strong relationship between marine algal vBPOs and vIPOs. This study will enhance our understanding of the biological characteristics and economic values of S. japonica species.

Three Gracilaria species, G. chouae, G. blodgettii, G. vermiculophylla and a close relative species, Gracilariopsis lemaneiformis which is now nominated as Gracilaria lemaneiformis, are the typically indigenous species which are important resources for the production of special proteins, phycobilisomes, special carbohydrates, and agar in China. In this study, de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time. Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G. lemaneiformis and other three Gracilaria species. Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species. The genes encoding allophycocyanin, phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G. lemaneiformis had close relationship to genus Gracilaria, as well as that within genus Gracilaria, G. chouae had closer relationship to G. vermiculophylla rather than to G. blodgettii. The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.

Brown algae (Chromista, Ochrophyta, Phaeophyceae) are a large group of multicellular algae that play important roles in the ocean’s ecosystem and biodiversity. However, poor molecular bases for studying their phylogenetic evolutions and novel metabolic characteristics have hampered progress in the field. In this study, we sequenced the de novo transcriptome of 18 major species of brown algae in China, covering six orders and seven families, using the high-throughput sequencing platform Illumina HiSeq 2000. From the transcriptome data of these 18 species and publicly available genome data of Ectocarpus siliculosus and Phaeodactylum tricornutum, we identified 108 nuclear-generated orthologous genes and clarified the phylogenetic relationships among these brown algae based on a multigene method. These brown algae could be separated into two clades: Clade Ishigeales-Dictyotales and Clade Ectocarpales-Laminariales-Desmarestiale-Fucales. The former was at the base of the phylogenetic tree, indicating its early divergence, while the latter was divided into two branches, with Order Fucales diverging from Orders Ectocarpales, Laminariales, and Desmarestiale. In our analysis of taxonomy-contentious species, Sargassum fusiforme and Saccharina sculpera were found to be closely related to genera Sargassum and Saccharina, respectively, while Petalonia fascia showed possible relation to genus Scytosiphon. The study provided molecular evidence for the phylogenetic taxonomy of brown algae.

Ishige okamurae, with leathery branched narrow fronds consisting of cylindrical hairs, is the typical species of the genus Ishige, which is considered as one of the most basal genera in the phylogeny of the Phaeophyceae. Apart from great public interest from the evolutionary respect, more attention has been brought on the abundant bioactive compounds in I. okamurae for therapeutic or economic considerations, such as diphlorethohydroxycarmalol and ishigoside. Yet little is known about related key genes or metabolic pathways involved in I. okamurae, which calls upon us to carry out global analyses of transcriptome by next generation sequencing. Altogether, we obtained 78 583 assembled scaffolds with N50 of 1 709 nucleotides, and 25 357 unigenes with significant BLAST matches (E-value cutoff of 10−5). In terms of characterization of the transcriptome of I. okamurae, we focused on anti-stress metabolic pathways and synthetic routes of bioactive compounds in an attempt to obtain a better understanding of the interactive organism-environment regulatory networks. Pathway-based analysis helped us to deepen our comprehension of the interaction between I. okamurae and its surroundings, with MAPK signal pathway as an example. Furthermore, we discovered a wide range of novel putative functional proteins that could be of wide application, such as Rab family, using sequence-based transcriptome. In conclusion, transcriptome characterization of I. okamurae (Phaeophyceae) shows strong environmental acclimation.

Species of Sargassum are distributed worldwide, and are of great ecological and economic importance in marine ecosystems and bioresources. In this study, transcriptome sequencings of six Sargassum species were performed for the first time using an Illumina platform. For each sample, a total of 2.1–2.5 Gb of nucleotides are collected and assembled into 69 871–116 790 scaffolds, with an average length of 410–550 bp and N50 length of 756–1 462 bp. A total of 20 512–28 684 unigenes of each sample were annotated and compared well with known gene sequences from nr database. Clusters of Orthologous Groups (COG), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were also performed for further understanding of gene functions and regulation pathways. Gene expression levels were calculated based on RPKM values and compared among these species, especially for those genes related to carbohydrate metabolism. Cluster analyses indicated that the differences of global gene expression between S. fusiforme, which was nominated as Hizikia fusiformis before, and other five species were not significant. Further phylogenetic analysis of 108 orthologous genes confirmed that S. fusiforme had closer relationship with S. hemiphyllum rather than S. horneri. These transcriptome data provided valuable information for better understanding of genome and gene characteristics of Sargassum algae and benefiting comparative and phylogenetic studies of Phaeophyceae species in future studies.

The chloroplast and mitochondrion of red algae (Phylum Rhodophyta) may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using red algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly available complete genomes of mitochondria and chloroplasts of Rhodophyta. We have found that red algae were divided into three clades of orders, Florideophyceae, Bangiophyceae and Cyanidiophyceae. Taxonomy resolution for Class Florideophyceae showed that Order Gigartinales was close to Order Halymeniales, while Order Gracilariales was in a clade of Order Ceramials. We confirmed Prionitis divaricata (Family Halymeniaceae) was closely related to the clade of Order Gracilariales, rather than to genus Grateloupia of Order Halymeniales as reported before. Furthermore, we found both mitochondrial and chloroplastic genes in Rhodophyta under negative selection (Ka/Ks < 1), suggesting that red algae, as one primitive group of eukaryotic algae, might share joint evolutionary history with these two organelles for a long time, although we identified some differences in their phylogenetic trees. Our analysis provided the basic phylogenetic relationships of red algae, and demonstrated their potential ability to study endosymbiotic events.

The chloroplast and mitochondrion of brown algae (Class Phaeophyceae of Phylum Ochrophyta) may have originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly available complete genomes of mitochondria and chloroplasts of Kingdom Chromista. We have found that there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others (Phylum Cryptophyta and Haptophyta) in Kingdom Chromista, and identified more diversity in chloroplast genes than mitochondrial ones in their phylogenetic trees. Taxonomy resolution for Class Phaeophyceae showed that it was divided into Laminariales-Ectocarpales clade and Fucales clade, and phylogenetic positions of Kjellmaniella crassifolia, Hizikia fusifrome and Ishige okamurai were confirmed. Our analysis provided the basic phylogenetic relationships of Chromista algae, and demonstrated their potential ability to study endosymbiotic events.