Protein-coding gene annotation. To search for homologous genes, the protein sequences from all ferns and lycophytes transcriptomes in the OneKP project 1 were retrieved and aligned to the A. capillus-veneris genome, using GeneWise 2 . For transcriptome-based prediction, nineteen transcriptomes covering the entire life cycle of A. capillus-veneris were generated in this study (Supplementary Table 8). RNA was extracted using the Qiagen RNeasy protocol and sequenced on an Illumina HiSeq 4000 with a 300 bp insert size. For transcriptome-based prediction, the HISAT2 3 and StringTie 4 programs were used for transcript assembly 5 . The program PASA (http://pasapipeline.github.io) was used to align spliced transcripts and annotate candidate genes. Ab initio prediction was performed with AUGUSTUS 6 , GlimmerHMM 7 , and SNAP 8 . Finally, nonredundant gene models were obtained with EVidenceModeler (version 1.1.0) 9 to integrate the gene models developed by different datasets.To validate the assembly quality, RNA-seq reads from nineteen tissues (Supplementary Table 8), together with publicly available EST sequences from the NCBI database (downloaded from http://togodb.dbcls.jp/library), were mapped to the A. capillus-veneris genome using HISAT2 3 and BLAT 10 with default parameters, respectively. The BLAT results were filtered with an identity and coverage cutoff of 0.9.Identification of noncoding RNAs. We used tRNAscan-SE (version 2.0rc2) 11 , with default parameters, to search for tRNAs in the A. capillus-veneris genome. A total of 1,624 tRNAs were found. Moreover, the Rfam14.0 database 12 , including 3,445 noncoding RNA families, was used to annotate additional noncoding RNAs (ncRNAs), including miRNAs, snRNAs, and tRNAs, using INFERNAL (version 1.1.2) 13 program.We predicted rRNA (5S, 5.8S, 28S, 18S) by using HMM searching based rRNA predicator Barrnap (version 0.9, https://github.com/tseemann/barrnap#barrnap), with default parameters. We finally identified 145 5S, 75 5.8S, 155 28S, and 165 18S sequences and their locations within the genome assembly of A. capillus-veneris.
Primary malignant melanoma of esophagus (PMME) is a rare malignant tumor of esophagus. This study aimed to investigate the clinic pathologic characteristics and analyze the factors that might affect the prognosis of PMME patients. A total of 20 PMME patients who underwent surgical treatment in our hospital from 1975 to 2017 were analyzed. The clinical data, surgical and pathologic features of all patients were collected. For 20 PMME patients, the average age was 57.3 ± 10.7 years, and the male patients account for 75%. Most of the tumors (95%) were located in the middle and lower of the esophagus. There were 7 patients with primary tumor invasion beyond the muscular layer (T3 + T4) and 10 patients with lymph node metastasis (LNM). The median survival time of 20 patients was 12 months, and the 1-year and 5-year survival rates were 50% and 16.9%, respectively. The probability of LNM in tumors confined to submucosa (T1) and myometrium (T2) was lower than that in tumors with deeper invasion (T3, T4) ( P = .035). Multivariate analysis showed that tumor node metastasis (TNM) staging was the independent prognostic factor for survival of PMME patients (hazard ratio [95% confidence interval], 4.15 [1.36–12.67]; P = .012). For PMME patients, tumors with deeper invasion were more likely to have LNM, and TNM staging was an independent predictor of prognosis for survival. Early detection of the disease and radical resection of the tumor are critical for better survival of the PMME patients.
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