Journal: eLife

Article Title: Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis

doi: 10.7554/eLife.22054

Figure Lengend Snippet: ( a ) Intracapsular algae (Population 1) were removed from intact eggs using a syringe and hypodermic needle (photo credit: Roger Hangarter). Embryos were decapsulated and washed, and the liver diverticulum region (dashed line), containing high concentrations of algae (red dots), was isolated and dissociated into a single cell suspension (illustration adapted from ). The dissociated cells were screened for A. maculatum endoderm cells without alga (black arrowheads) and endoderm cells with intracellular alga (green arrowhead). Scale bars on microscope images are 20 µm. ( b ) Isolated endoderm cell, and isolated endoderm cell with intracellular alga. Scale bars on microscope images are 20 µm. ( c ) Representative cDNA distribution (bioanalyzer trace) from a population of 50 manually isolated A. maculatum endoderm cells. Peaks at 35 bp and 10380 bp are markers. Due to evidence of lysed A. maculatum cells observed in the cell suspension fluid after dissociation of A. maculatum embryos (debris seen in dissociated A. maculatum microscope images in ( a ) and ( b )), that fluid was tested for the presence of contaminating mRNA. mRNA was not detected in the surrounding fluid, . Lower limit abundance thresholds , and correction for low sequencing depth in intracelluar algal samples were implemented to obtain the final gene sets used for differential expression analysis. Depth of sequencing was not biased for A. maculatum cell with and without alga samples . Library preparation GC bias affected the completeness of the algal transcriptome obtained from intracapsular and intracellular O. amblystomatis . ( d and e ) Dotplots of log 2 fold change vs. expression level. The blue horizontal lines are plus and minus 4-fold change in expression between samples. The red dots are genes with FDR adjusted p-values<0.05, indicating a significant difference in expression level between conditions. ( d ) Differentially expressed algal transcripts. ( e ) Differentially expressed salamander transcripts. DOI: http://dx.doi.org/10.7554/eLife.22054.003 10.7554/eLife.22054.004 Figure 1—source data 1. Raw counts matrix with counts for all reads mapped to the total evidence assembly (the assembly of all salamander and algal reads from wild-collected samples). The data in this file (after filtering and normalization) was used to generate the dotplots in , – , and . This is the raw data that was used for differential expression analysis. Rows are genes. Column names are as follows: S2a-S5a are counts for salamander cells without algae. S2b-S5b are counts for salamander cells with intracellular algae (samples are paired from the same individuals, such that S2a and S2b came from the same salamander). A1-A3 are intracapsular algae samples. RK_* are cultured algal samples. DOI: http://dx.doi.org/10.7554/eLife.22054.004 10.7554/eLife.22054.005 Figure 1—source data 2. List of 6,726 algal gene IDs used in differential expression analysis. Use to filter raw counts matrix to get final algal gene list. DOI: http://dx.doi.org/10.7554/eLife.22054.005 10.7554/eLife.22054.006 Figure 1—source data 3. List of 46,549 salamander gene IDs used in differential expression analysis. Use to filter raw counts matrix to get final salamander gene list. DOI: http://dx.doi.org/10.7554/eLife.22054.006

Article Snippet: Blue bars represent transcripts found only in the transcriptome assembly from the TrueSeq library preparation method, that are absent from the transcriptome generated using the SMARTer cDNA synthesis-Nextera-XT library preparation method.

Techniques: Algae, Isolation, Suspension, Microscopy, Sequencing, Quantitative Proteomics, Expressing, Cell Culture

Journal: eLife

Article Title: Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis

doi: 10.7554/eLife.22054

Figure Lengend Snippet: ( a ) Representative cDNA distribution (bioanalyzer trace) from a population of 50 manually isolated A. maculatum endoderm cells. ( b ) No cDNA was produced when the fluid the cells were suspended in was tested indicating that the cDNA populations from manually isolated A. maculatum endoderm cells was specific and not contaminated with cDNAs derived from randomly lysed cells. In both ( a ) and ( b ), the peaks at 35 bp and 10380 bp are markers. DOI: http://dx.doi.org/10.7554/eLife.22054.007

Article Snippet: Blue bars represent transcripts found only in the transcriptome assembly from the TrueSeq library preparation method, that are absent from the transcriptome generated using the SMARTer cDNA synthesis-Nextera-XT library preparation method.

Techniques: Isolation, Produced, Derivative Assay

Journal: eLife

Article Title: Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis

doi: 10.7554/eLife.22054

Figure Lengend Snippet: ( a ) The GC content distribution of algal transcripts generated using TrueSeq library preparation of total RNA, sequenced on the MySeq platform with approximately 30 million 75 bp paired end reads. 79% of eukaryote BUSCOs were detected in this assembly. The median GC content (green dashed line) is 62%. ( b ) The GC content distribution from ( a ), split by library preparation method. Red bars represent algal transcripts found in transcriptomes generated by both library preparation methods (SMARTer-Netxtera-XT and TruSeq). Blue bars represent transcripts found only in the transcriptome assembly from the TrueSeq library preparation method, that are absent from the transcriptome generated using the SMARTer cDNA synthesis-Nextera-XT library preparation method. There is an apparent bias against high GC content algal transcripts in library prepared using the SMARTer cDNA synthesis-Nextera-XT protocol (Kolgomorov-Smirnov test, p<2.2 × 10 −16 ). Both libraries were sequenced to a similar depth of approximately 30 million reads for the alga-only samples in the total-evidence assembly from the SMARTer-cDNA synthesis-Nextera-XT library and 30 million reads for the TrueSeq library from unialgal cultures. Since sequencing depth was equivalent and GC bias is apparent, the data suggests that GC bias in the SMARTer-cDNA synthesis-Nextera-XT library is what accounts for the low number of detected BUSCOs (49%) in the algal transcriptome generated from wild-collected algal samples associated with salamander eggs and cells. (C.) The distribution of GC content in A. maculatum transcripts (gray bars) is centered around much lower GC content transcripts (median GC content of 43%) compared to that of O. amblystomatis (green bars, median GC content of 62%). The A. maculatum assembly contained 88% of eukaryote BUSCOs. Our evidence points to bias against high GC content transcripts in the SMARTer cDNA synthesis and Nextera-XT library prep method, that becomes significant above 60% GC content. Transcripts with GC content of 60% or greater are in the tail of the salamander GC content distribution, but near the median of the algal GC content distribution. This offers an explanation for the BUSCO results, where the salamander transcriptome from the wild-collected samples is comprehensive, while the algal transcriptome from the same samples and library prep methods is missing around 40% of the algal transcriptome. DOI: http://dx.doi.org/10.7554/eLife.22054.011

Article Snippet: Blue bars represent transcripts found only in the transcriptome assembly from the TrueSeq library preparation method, that are absent from the transcriptome generated using the SMARTer cDNA synthesis-Nextera-XT library preparation method.

Techniques: Generated, cDNA Synthesis, Sequencing

Journal: eLife

Article Title: Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis

doi: 10.7554/eLife.22054

Figure Lengend Snippet: Red lines indicate the GC content or transcript length biases in reads obtained from SMARTer-cDNA synthesis-Nextera-XT libraries. Blue lines indicate the GC content or transcript length biases in reads obtained from TrueSeq libraries. ( a ) GC content and length are plotted against ‘QRfit’ which is a measure of fit by quantile regression to the models in . This metric approximates bias in the sequence dataset by comparing read counts to expected models based on quantiles in the distribution of the GC content of the transcripts. The opposing trends in the two sets of lines shows that GC content bias between the two different libraries is vastly different. The reads obtained from SMARTer-cDNA synthesis-Nextera-XT libraries will tend to have more counts for low GC content transcripts, while the reads obtained from TrueSeq libraries will tend to have more counts for high GC content transcripts, systemically. ( b ) There is also some moderate transcript length bias differences between the two library prep methods visualized as the separation between the groups of red and blue lines. The methods implemented by the conditional quantile normalization (cqn) package in R handles both types of bias to make the gene count data from both library preparation methods comparable. DOI: http://dx.doi.org/10.7554/eLife.22054.026

Article Snippet: Blue bars represent transcripts found only in the transcriptome assembly from the TrueSeq library preparation method, that are absent from the transcriptome generated using the SMARTer cDNA synthesis-Nextera-XT library preparation method.

Techniques: cDNA Synthesis, Sequencing

Journal: eLife

Article Title: Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis

doi: 10.7554/eLife.22054

Figure Lengend Snippet: O. amblystomatis qPCR primer sequences. Primer pairs for four reference genes ( RACK1, YPTC1, RPL32, H2B1 ), and five response genes ( PhT1.2, NaPhT1 [ANTR1], AMT1.2, NRT2.4, DUR3 ) used in this study. Efficiency values were measured per amplicon using a standard curve with five two-fold dilutions of cDNA. DOI: http://dx.doi.org/10.7554/eLife.22054.027

Article Snippet: Blue bars represent transcripts found only in the transcriptome assembly from the TrueSeq library preparation method, that are absent from the transcriptome generated using the SMARTer cDNA synthesis-Nextera-XT library preparation method.

Techniques: Amplification, Sequencing