GigaScience, 6, 2017, 1–18 doi: 10.1093/gigascience/gix057 Advance Access Publication Date: 13 July 2017 Research RESEARCH Transcriptome analysis of the response of Burmese python to digestion Jinjie Duan1,∗, Kristian Wejse Sanggaard2,3, Leif Schauser4, Sanne Enok Lauridsen5, Jan J. Enghild2,3, Mikkel Heide Schierup1,5,∗ and Tobias Wang5,∗ 1Bioinformatics Research Center, Aarhus University, C.F. Moellers Alle 8, Aarhus C, Denmark, 2Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, Aarhus C, Denmark, 3Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, Aarhus C, Denmark, 4QIAGEN Aarhus, Silkeborgvej 2, Aarhus C, Denmark and 5Department of Bioscience, Aarhus University, Ny Munkegade 116, Aarhus C, Denmark ∗Correspondence address. Jinjie Duan, Bioinformatics Research Center, Aarhus University, C.F. Moellers Alle 8, Aarhus C, Denmark. Tel: +45 87168497; Fax: +45 87154102; E-mail: [email protected]; Mikkel Heide Schierup, Bioinformatics Research Center, Aarhus University, C.F. Moellers Alle 8, Aarhus C, Denmark. Tel: +45 87156535; Fax: +45 87154102; E-mail: [email protected]; Tobias Wang, Department of Bioscience, Aarhus University, Ny Munkegade 116, Aarhus C, Denmark. Tel: +45 87155998; Fax: +45 87154326; E-mail: [email protected] Abstract Exceptional and extreme feeding behaviour makes the Burmese python (Python bivittatus) an interesting model to study physiological remodelling and metabolic adaptation in response to refeeding after prolonged starvation. In this study, we used transcriptome sequencing of 5 visceral organs during fasting as well as 24 hours and 48 hours after ingestion of a large meal to unravel the postprandial changes in Burmese pythons. We first used the pooled data to perform a de novo assembly of the transcriptome and supplemented this with a proteomic survey of enzymes in the plasma and gastric fluid. We constructed a high-quality transcriptome with 34 423 transcripts, of which 19 713 (57%) were annotated. Among highly expressed genes (fragments per kilo base per million sequenced reads > 100 in 1 tissue), we found that the transition from fasting to digestion was associated with differential expression of 43 genes in the heart, 206 genes in the liver, 114 genes in the stomach, 89 genes in the pancreas, and 158 genes in the intestine. We interrogated the function of these genes to test previous hypotheses on the response to feeding. We also used the transcriptome to identify 314 secreted proteins in the gastric fluid of the python. Digestion was associated with an upregulation of genes related to metabolic processes, and translational changes therefore appear to support the postprandial rise in metabolism. We identify stomach-related proteins from a digesting individual and demonstrate that the sensitivity of modern liquid chromatography/tandem mass spectrometry equipment allows the identification of gastric juice proteins that are present during digestion. Keywords: Burmese python; transcriptome; tissue expression; digestion; pathway; proteome Background lar intervals. The Burmese python is an iconic example of this extreme phenotype [1]. Many species of pythons easily endure All animals exhibit dynamic changes in the size and functional months of fasting, while remaining capable of subduing and in- capacities of bodily organs and tissues to match energetic main- gesting very large meals. In Burmese pythons, digestion is at- tenance costs to prevailing physiological demands [1]. This phe- tended by a large and rapid rise in mass and/or functional ca- notypic flexibility is particularly pronounced in the digestive or- pacity of the intestine, stomach, liver, heart, and kidneys [2–4] gans in animals that naturally experience prolonged periods of in combination with a stimulation of secretory processes and an fasting but are capable of ingesting large prey items at irregu- Received: 23 November 2016; Revised: 12 April 2017; Accepted: 6 July 2017 C The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 2 Duan et al. Figure 1: The workflow of Python RNA-Seq data analysis. The diagram shows the main steps and bioinformatics tools used inthestudy. activation of enzymes and transporter proteins. These physio- proaches and opted instead to use our high-coverage data to logical responses are associated with a many-fold rise in aerobic build a de novo transcriptome assembly to identify differentially metabolism. Hence, the Burmese python is an excellent model expressed genes (DEGs). To identify the enzymes involved in the to study the mechanisms underlying extreme metabolic transi- digestion process, we isolated the digestive fluid and charac- tions and physiological remodelling in response to altered de- terized the protein composition using a proteomics-based ap- mand [1, 3, 5–10]. proached. This also allowed us to identify the major hydrolytic The postprandial changes in the morphology and physiol- enzymes used to digest the large and un-masticated meals. ogy of the intestine, heart, and other organs have been de- scribed in some detail in pythons [1, 5, 8, 9, 11], but only a few studies [12–14] have addressed the underlying transcrip- Analyses tional changes of this interesting biological response. Transcrip- Data summary tome sequencing technology now allows comprehensive sur- veys [15, 16], prompting our use of transcriptome sequencing A total of 277 485 924 raw paired reads (2∗101 bp, insert size of the heart, liver, stomach, pancreas, and intestine in snakes 180 bp) were obtained from Illumina Hi-Seq 2000 sequenc- that had fasted for 1 month, at 24 and 48 hours into the post- ing of 15 non-normalized cDNA libraries derived from 5 tis- prandial period. These organs were chosen because a number of sues (heart, liver, stomach, pancreas, and intestine) at 3 time earlier studies have revealed their profound phenotypic changes points (fasted for 1 month, 24 hours, and 48 hours postpran- during the postprandial period [1–4, 17], and they are therefore dial) and 10 DSN-normalized cDNA libraries (see the Methods likely to exhibit large changes in gene expression. Differential section) (Supplementary Table S1). After removal of low-quality gene expression in some of these organs has previously been reads (see the Methods section), 213 806 111 (77%) high-quality reported [12–14], but we provide new data on 48 hours into the paired reads were retained. These reads contained a total of digestive period and the first descriptions of gene expression in 43 146 073 200 bp nucleotides with a mean Phred quality higher the stomach and the pancreas. As the Burmese python refer- than 37 (Q37). To develop a comprehensive transcriptomics re- ence genome assembly [12] is relatively fragmented (contig size source for the Burmese python (Fig. 1), we pooled these high- N50 ∼10 kb), we found it impractical to use re-sequencing ap- quality reads from 25 libraries for subsequent de novo assembly. Transcriptome of Burmese python 3 Table 1: Summary of transcriptome assembly of Burmese python. These results are consistent with a survey [20] of assessment completeness of 28 transcriptomes from 18 vertebrates. In this Parameter De novo assembly survey, most of the transcriptomes from species with close phy- logenetic relationships to snakes contain less than 50% com- Total transcripts 34 423 plete BUSCOs and more than 40% missing BUSCOs. Therefore, Annotated transcripts with nr NCBI 19 713 we conclude that the quality of our transcriptome assembly was Annotated transcripts with GO term 16 992 Minimum transcript size (nt) 100 acceptable. Medium transcript size (nt) 605 Mean transcript size (nt) 1034 Transcriptome annotation Largest transcript (nt) 26 010 N50 6240 A total of 19 713 transcripts (57% of 34 423) were annotated using N50 size (nt) 1673 transfer of blastx hit annotation against the non-redundant (nr) Total assembled bases (Mb) 35.6 NCBI peptide database [21]. To assign proper annotation for each transcript, we chose the first best hit that was not represented in uninformative descriptions (Supplementary Table S4). The most closely related species with an annotated genome, Anolis caroli- De novo transcriptome assembly and evaluation nensis, was able to annotate 10 704 transcripts (54% of all anno- tated transcripts). Burmese Python and Anolis carolinensis both As short k-mers have a higher propensity to generate misas- belong to the reptilian order Squamata and diverged from each sembled transcripts when using a de Bruijn graph–based de novo other approximately 160 million years ago [22]. assembler, such as Velvet [18], we conservatively chose an as- Blast2GO was used to annotate these 19 713 transcripts [23], sembly generated using long k-mers for subsequent analysis, at of which 16 992 could be assigned to 1 or more gene ontology the cost of some sensitivity regarding assembled isoforms. Thus, (GO) terms and their putative functional roles. The distributions balancing key metrics (Supplementary Table S2), we used an of the most frequently identified GO term categories for biolog- assembly based on the longest k-mer (= 95) (Table 1)asithad ical processes, molecular function, and cellular component are the fewest scaffolds/transcripts (34 423) but represented a very shown in Fig.