BackgroundRestriction site associated DNA sequencing (RADseq) has the potential to be a broadly applicable, low-cost approach for high-quality genetic linkage mapping in forest trees lacking a reference genome. The statistical inference of linear order must be as accurate as possible for the correct ordering of sequence scaffolds and contigs to chromosomal locations. Accurate maps also facilitate the discovery of chromosome segments containing allelic variants conferring resistance to the biotic and abiotic stresses that threaten forest trees worldwide. We used ddRADseq for genetic mapping in the tree Quercus rubra, with an approach optimized to produce a high-quality map. Our study design also enabled us to model the results we would have obtained with less depth of coverage.ResultsOur sequencing design produced a high sequencing depth in the parents (248×) and a moderate sequencing depth (15×) in the progeny. The digital normalization method of generating a de novo reference and the SAMtools SNP variant caller yielded the most SNP calls (78,725). The major drivers of map inflation were multiple SNPs located within the same sequence (77% of SNPs called). The highest quality map was generated with a low level of missing data (5%) and a genome-wide threshold of 0.025 for deviation from Mendelian expectation. The final map included 849 SNP markers (1.8% of the 78,725 SNPs called). Downsampling the individual FASTQ files to model lower depth of coverage revealed that sequencing the progeny using 96 samples per lane would have yielded too few SNP markers to generate a map, even if we had sequenced the parents at depth 248×.ConclusionsThe ddRADseq technology produced enough high-quality SNP markers to make a moderately dense, high-quality map. The success of this project was due to high depth of coverage of the parents, moderate depth of coverage of the progeny, a good framework map, an optimized bioinformatics pipeline, and rigorous premapping filters. The ddRADseq approach is useful for the construction of high-quality genetic maps in organisms lacking a reference genome if the parents and progeny are sequenced at sufficient depth. Technical improvements in reduced representation sequencing (RRS) approaches are needed to reduce the amount of missing data.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3765-8) contains supplementary material, which is available to authorized users.
Out of 83 human colorectal and 44 stomach cancers transplanted subcutaneously in nude mice, tumor take was observed in 78 and 68 %, respectively. Progressive tumor growth (product of two tumor diameters . > 60 mm2 after 90 days) was found in 49 and 32%. Serial passage was done in 46 colorectal and 17 stomach cancers. In 5 out of 6 esophageal cancers tumor take was found, and serial passage was performed in 4 tumors. Tumor stage was the most important factor for the take rate. Metastasized or locally advanced tumors of the large bowel and stomach were grown in 89 and 54%, respectively, which was significantly higher than in the localized stage. The take rate was independent of the degree of differentiation, the amount of fibrous tissue, CEA content, sex and tumor localization, except for tumors originating in the cardia in comparison to other parts of the stomach. The similarity of the xenografts in serial passage in comparison to the donor tumor was shown by histological and immunological (CEA) examinations. The presence of human isoenzymes such as esterase-D and LDH demonstrate the human origin of the tumors. However, most of the xenografts were growing more rapidly in the serial passage than in early passages. 31 comparisons of tumor response in the patient and in nude mice were made in 20 different tumors. The highly correct prediction rate for resistance (100%) and sensitivity (86%) validates the xenograft system. 34 colorectal, 14 stomach and 3 esophageal cancers were selected and characterized as tumor models. This tumor panel is continuously available for therapeutic and biological studies. For surgical purposes the xenografts can also be grown in athymic nude rats. The most relevant model for the clinic is available if the human tumors are implanted at the site of tumor origin.
Recently, the 5‐year results of a multicenter trial on proximal gastric vagotomy (PGV) have been reported. Symptomatic results in 415 duodenal ulcer patients, according to the Visick grading, were as follows: grade 1, 64%; 2, 28%; 3, 4%; and 4, 4%. It appears that one‐third of the patients are symptomatic, and the question arises as to which standard such a Visick grade pattern should be compared. Five hundred sixty‐one healthy controls (blood donors), without history of peptic ulcer or previous gastric operation, have been examined according to a standard questionnaire identical to that used for follow‐up interrogation in the PGV trial. The control group was matched for sex and age with the trial population. Answers to 3 screening questions were compared with the result of detailed interrogation. The control group's Visick pattern was as follows: grade 1, 64.5%; 2, 28.9%; 3, 6.4%; and 4, 0.2%. Symptoms most frequently encountered were those of dyspepsia (pain, epigastric fullness) and gastroesophageal reflux. Dumping (2%) and diarrhea (4%) were also noted. Our conclusions are these: (1) The Visick grade pattern 5 years after PGV is almost identical with that of healthy controls. (2) Dull pain, epigastric fullness, and reflux symptoms are not specific signs after PGV. (3) Screening questions are misleading and follow‐up examinations must be based on a standard questionnaire to provide valid information. (4) The separation between Visick grade 1 and 2 should be abandoned, as it has no clinical importance. (5) PGV has virtually no specific long‐term sequelae except recurrence.
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