Are special read alignment strategies necessary and cost-effective when handling sequencing reads from patient-derived tumor xenografts?

Background

Patient-derived tumor xenografts in mice are widely used in cancer research and have become important in developing personalized therapies. When these xenografts are subject to DNA sequencing, the samples could contain various amounts of mouse DNA. It has been unclear how the mouse reads would affect data analyses. We conducted comprehensive simulations to compare three alignment strategies at different mutation rates, read lengths, sequencing error rates, human-mouse mixing ratios and sequenced regions. We also sequenced a nasopharyngeal carcinoma xenograft and a cell line to test how the strategies work on real data.

Results

We found the "filtering" and "combined reference" strategies performed better than aligning reads directly to human reference in terms of alignment and variant calling accuracies. The combined reference strategy was particularly good at reducing false negative variants calls without significantly increasing the false positive rate. In some scenarios the performance gain of these two special handling strategies was too small for special handling to be cost-effective, but it was found crucial when false non-synonymous SNVs should be minimized, especially in exome sequencing.

Conclusions

Our study systematically analyzes the effects of mouse contamination in the sequencing data of human-in-mouse xenografts. Our findings provide information for designing data analysis pipelines for these data.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1172) contains supplementary material, which is available to authorized users.     

The importance of the lysophosphatidylcholine and choline moiety of bile phosphatidylcholine in lymphatic transport of fat

A luminal supply of biliary phosphatidylcholine is important in the translocation of absorbed fat into lymph and in the amount and composition of phosphatidylcholine concurrently synthesized. This study was undertaken to determine whether the effect was due to absorbed lysophosphatidylcholine, to a specific (1-palmitoyl) biliary lysophosphatidylcholine or to extra choline supplied by lysophosphatidylcholine. Rats with bile fistulae and thoracic duct lymph fistulae were given test meals of oleic acid and monoolein (molar ratio 2 : 1) infused duodenally for 8 h. Addition of choline chloride to the test meal increased lymphatic output of triglyceride and phospholipid but not to values found previously in rats with supplements of bile phosphatidylcholine or with bile ducts intact. Addition of dioleoyl phosphatidylcholine increased triglyceride and phospholipid output to values found in rats with intact bile ducts. Since dioleoyl phosphatidylcholine was as efficient as biliary phosphatidylcholine it was concluded that a luminal supply of 1-palmitoyl lysophosphatidylcholine was not essential. It seemed likely from the smaller effect of supplemented choline and from the fatty acid composition of lymph phosphatidylcholine that the essential requirement was a supply of absorbed lysophosphatidylcholine for rapid reacylation to phosphatidylcholine.     

Structure of human apolipoprotein A-IV: a distinct domain architecture among exchangeable apolipoproteins with potential functional implications

Apolipoprotein A-IV (apoA-IV) is an exchangeable apolipoprotein that shares many functional similarities with related apolipoproteins such as apoE and apoA-I but has also been implicated as a circulating satiety factor. However, despite the fact that it contains many predicted amphipathic alpha-helical domains, relatively little is known about its tertiary structure. We hypothesized that apoA-IV exhibits a characteristic functional domain organization that has been proposed to define apoE and apoA-I. To test this, we created truncation mutants in a bacterial system that deleted amino acids from either the N- or C-terminal ends of human apoA-IV. We found that apoA-IV was less stable than apoA-I but was more highly organized in terms of its cooperativity of unfolding. Deletion of the extreme N and C termini of apoA-IV did not significantly affect the cooperativity of unfolding, but deletions past amino acid 333 on the C terminus or amino acid 61 on the N terminus had major destabilizing effects. Functionally, apoA-IV was less efficient than apoA-I at clearing multilamellar phospholipid liposomes and promoting ATP-binding cassette transporter A1-mediated cholesterol efflux. However, deletion of a C-terminal region of apoA-IV, which is devoid of predicted amphipathic alpha helices (amino acids 333-376) stimulated both of these activities dramatically. We conclude that the amphipathic alpha helices in apoA-IV form a single, large domain that may be similar to the N-terminal helical bundle domains of apoA-I and apoE but that apoA-IV lacks the C-terminal lipid-binding and cholesterol efflux-promoting domain present in these apolipoproteins. In fact, the C terminus of apoA-IV appears to reduce the ability of apoA-IV to interact with lipids and promote cholesterol efflux. This indicates that, although apoA-IV may have evolved from gene duplication events of ancestral apolipoproteins and shares the basic amphipathic helical building blocks, the overall localization of functional domains within the sequence is quite different from apoA-I and apoE.     

Mechanism of the induction of brain c-Fos-positive neurons by lipid absorption

Many gastrointestinal meal-related signals are transmitted to the central nervous system via the vagus nerve and thereby control changes in meal size. The c-Fos-positive neuron has been used as a marker of neuronal activation after lipid meals to examine the contribution of a selective macronutrient on brain neurocircuit activity. In rats fed Intralipid, the c-Fos-positive neurons were highly stimulated in the nucleus of the solitary tract (NTS) and in the hypothalamus, including the paraventricular nucleus (PVN), arcuate nucleus of the hypothalamus (ARC), and ventromedial hypothalamus at 4 h lipid feeding. However, c-Fos-like immunoreactivity was markedly attenuated in these brain regions when chylomicron formation/secretion was blocked by Pluronic L-81. After lymph was diverted from the lymph cannulated animals, the rats had a lower number of c-Fos-positive cells in the NTS and ARC. In contrast, the rats had higher c-Fos-positive neurons in PVN. The present study also revealed that c-Fos-positive neurons induced by feeding of Intalipid were abolished by CCK type 1 receptor antagonist, Lorglumide. We conclude that the formation and/or secretion of chylomicron are critical steps for initiating neuronal activation in the brain.     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Effect of fat pre-feeding on bile flow and composition in the rat

Studies were conducted in rats to determine if the increase in lymph triacylglycerol output on pre-feeding a 20% glyceryltrioleate diet (Mansbach, C.M., II and Arnold, A. (1986) Am. J. Physiol. 251, G263-269) was due to an increase in phosphatidylcholine output into bile. Rats who were fed chow or pre-fed the 20% fat diet were equipped with biliary and duodenal cannulas and infused with glucose-saline while bile was collected hourly. The next day a taurocholate-glyceryltrioleate infusion was given and bile collected for 5 h. Bile flow, bile acid, phosphatidylcholine and cholesterol output were greater in the chow fed group than controls during the 6 h of the glucose saline period. Outputs were low overnight. During the taurocholate-glyceryltrioleate infusion, bile flow, bile acid, phosphatidylcholine and cholesterol output were all greater in the fat pre-fed group than the chow fed controls. We conclude that fat pre-feeding profoundly influences biliary composition and flow. The 2-fold increase in biliary phosphatidylcholine output during duodenal lipid infusion offers a potential explanation for the increased delivery of triacylglycerol into the lymph in rats on a similar fat pre-feeding program.     

Effect of Photoperiod and End-of-Day Light Quality on Alkaloids and Phenolic Compounds of Tobacco

Tobacco plants (Nicotiana tabacum L.) were grown on long or short photoperiods followed by 5 minutes of red or far red radiation each day. Plants that received 16-hour photoperiods had a significantly higher concentration of total alkaloids and total phenolics than those that received 8-hour photoperiods. Significantly higher total alkaloid content was found in plants that received red rather than far red radiation last each day. Within each photoperiod, plants that received far red had higher concentrations of soluble phenols, particularly of chlorogenic acid. The interactions among these variables upon alkaloid and phenolic contents are discussed.     

Phylogeography of the giant wood spider (Nephila pilipes, Araneae) from Asian–Australian regions

Aim There are currently few population genetic studies on widely distributed SE Asian terrestrial organisms. We have studied the genetic diversification pattern of the giant wood spider, Nephila pilipes (Araneae: Tetragnathidae) to see whether fluctuations in rain forest extents generated by Quaternary climatic changes left signatures on populations of this agile terrestrial arthropod. Location The collecting localities were distributed in the following seven regions: (1) N Australia; (2) India (Calcutta, Karziranga and Sukna); (3) SE Asia (N Vietnam, Malaysia, Singapore and Bali); (4) SE China (Fujian, Guandong, Hong Kong and Hainan); (5) SW China (Guangxi and Yunnan); (6) E Asian islands (Ryukyu islands and Taiwan); and (7) the Philippine Islands. Methods A total of 374 specimens were collected from the East Asian continent and islands, SE Asia, India, and northern Australia. Mitochondrial cytochrome oxidase I gene partial sequences were used as the molecular marker to infer the phylogeographic diversification patterns. Results From the specimens collected, 67 haplotypes were identified, which could be grouped into five major clades. The dominant clade contained populations in regions ranging from Okinawa to Bali (spanning a distance of more than 4000 km), but their genetic variations were not structured and were not significantly associated with geographical distances. Three clades contained specimens collected from peripheral regions of the distribution range of N. pilipes, such as India, N Australia, and NE Asia. Members of the clade distributed in NE Asia were sympatric but those of the clades distributed in Australia and India were allopatric with those of the dominant clade. Main conclusions The results of this study indicate that, during Quaternary glacial periods, the rain forests in SE Asia might have been more or less continuous and thus generated an unstructured genetic diversification pattern of N. pilipes inhabiting this region. However, during such periods, populations in peripheral regions such as India, N Australia and NE Asia might have been isolated in refugia, thus accounting for the observed genetic divergence from populations in the SE Asian region.