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/ Developmental Biology 426 (2017) 143–154
quantification on a Synergy plate reader. Samples were then
pooled and sequenced with 72 bp reads with the Illumina HiSeq
Rapid chemistry.
The raw read data and annotated gene expression measures are
available in the Gene Expression Omnibus (accession number
GSE78034).
2.3. RNA-seq data analysis
The RNA-seq data consisted of three biological replicate sam-
ples from each stage (with the exception of stages 1 and 11, which
only had two replicates), for a total of 49 samples. RNA-seq data
were comprised of paired-end reads with a length of 101 bases
(polyAþ data) and single end reads with a length of 72 bases (total
RNA data). Adapter sequences were trimmed from reads using an
in-house script (available at www.axolomics.org/default/files/ffq.
ts.pe.ao.pl.tgz). Approximately 0.25% of reads were discarded for
having fewer than 28 bases after adapter trimming. No other
quality filters were applied. After filtering, the mean number of
sequenced read pairs per sample was " 31.5 million. For each
stage, the filtered reads from all replicates of that stage were
combined and assembled using Trinity (Grabherr et al., 2011)
(v2014-04-13p1, with parameters “–glue_factor 0.01 –min_iso_
ratio 0.1”). We chose not to build one single assembly using contigs
from all stages because of computational memory limitations and
concerns about creating “in-silico” transcript isoforms uniting
contigs from different developmental stages. After building the
stage-specific assemblies, a single combined non-redundant
transcriptome assembly was created by clustering all contigs from
all stage assemblies using USEARCH (Edgar, 2010) (v7.0.1090, with
parameters “-cluster_smallmem -id 0.95 -strand both”) and only
selecting the “centroid” contig from each cluster. The resulting
assembly had 896,365 contigs.
Quantification of each RNA-seq sample was then performed
using RSEM v1.1.6 (Li and Dewey, 2011) using the combined
transcriptome assembly as the reference database. RSEM was run
with the default options for paired-end data except for the “–no-
polyA” option, which was used because the assembled contigs
were not guaranteed to include polyAþ tails. By default, RSEM
uses the Bowtie aligner (Langmead et al., 2009) to map the reads
against the contigs and we had Bowtie v0.12.1 installed for this
purpose. RSEM employs an expectation maximization algorithm,
so that for reads that match to multiple contigs, RSEM assigns a
fraction of each read to each contig based on estimated abun-
dances of contigs based on unique reads (Li and Dewey, 2011).
To enable functional interpretation of the resulting transcrip-
tional profiles for the combined axolotl transcriptome assembly,
we used a comparative RNA-seq approach developed previously
for analysis of axolotl RNA-seq data (Stewart et al., 2013). Briefly,
the contigs of the combined assembly were first mapped to tran-
scripts from another species by running BLAST (NCBI BLAST
v2.2.18) with either the RefSeq RNA (via BLASTN), or protein (via
BLASTX) sequences for that species as the database and each
contig as a query. Contigs were assigned to transcripts by taking
the best BLAST hit with E-value o10 # 5. For each sample, the
expected fragment counts for each contig (as computed by RSEM),
were then converted to comparative transcript counts by summing
the fragment counts of contigs mapped to the same transcript.
Similarly, gene-level counts were obtained by summing the frag-
ment counts of transcripts that were annotated with the same
gene symbol. Thus, if two contigs map to different isoforms of the
same human gene or two contigs representing two axolotl in-
paralogs map to the same human gene, their counts will be
combined into the count for one human gene. Relative abun-
dances, in terms of transcripts per million (TPM), for genes were
computed by first normalizing each gene's fragment count by the
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sum of the “effective lengths” (length less the read length) of the
contigs mapped to that gene and then scaling the resulting values
such that they summed to one million over all genes. Contigs
mapping to rRNA transcripts and their respective counts were
removed from the analysis. The list of assembled and annotated
contigs is available at www.axolomics.org//sites/default/files/Ax-
oDevelTimecourse_Annotated_Contigs_with_Gene_Symbol.fa.tgz.
This comparative approach was run using both human and Xeno-
pus tropicalis as reference species. The NCBI RefSeq sets used for
human and X. tropicalis were dated 12/7/2009 and 8/3/2015, re-
spectively. It should be noted that by virtue of matching to human
(or frog) annotation there will be axolotl-specific genes that our
methodology a priori cannot capture.
We also estimated gene specific within-condition mean and
variance using median-by-ratio normalized expected counts
(normalized ECs) (Leng et al., 2013) of samples within each of the
17 stages. For each stage, the global mean-variance relationship is
represented by a fitted curve using all estimates in this stage. The
curve was fitted using polynomial regression on log(variance) " log
(mean), with 2 degrees of freedom.
2.4. Differentially expressed genes (DEGs)
We used the EBSeq package (Leng et al., 2013) to assess the
probability of a gene being differentially expressed between two
stages or two clusters. Given any two conditions, the false dis-
covery rate (FDR) is calculated based on the replicate or triplicate
samples. We also calculated the normalized ECs for each condition
(using the EBSeq method). We required that DEGs should have
FDR o5% and 42 fold-change of average normalized ECs be-
tween any given two conditions.
In the case of our comparative analysis of Xenopus develop-
ment, we acquired published data from Owens et al. (2016). We
used their calculated gene expression values (Gaussian process
lower median of'Transcripts per Embryo’/10000) for each stage.
See Owens et al. (2016) for details. We only used gene expression
values from the same stages as our axolotl data. We then defined
DEGs as genes that showed a greater than two-fold change of
(‘expression value’ þ1).
To compare HOX genes between axolotl and Xenopus (Owens
et al., 2016), we used a log10 transformed “expression measure þ1″
method (Chaudhuri et al., 2011; Shalek et al., 2014). The addition
of a value of 1 to'expression measure’ avoids undefined arithmetic
and suppresses reporting of high fold-ratio expression changes for
genes with relatively low expression in one or both sample groups.
2.5. Gene ontology analysis
Gene ontology (GO) analysis was performed using the R/allez
package (Newton et al., 2007) with each of 52 sets of DEGs (16
stage-to-stage analyses, 6 cluster-to-cluster analyses, 4 long time
interval stage-to-stage comparisons, with one set each of up-
regulated and down-regulated DEGs for each analysis). Only GO
terms with 50-800 associated genes were considered. GO terms
with Benjamini-Hochberg adjusted p values less than 0.01 were
considered as enriched.
2.6. Hierarchical clustering analysis
We clustered the stages based on correlations of their gene
expression vectors. To do so, we calculated the normalized ECs
(Leng et al., 2013) for each sample. Then, for each stage we aver-
aged the normalized ECs of each gene across all replicates from
that stage. We then calculated, for each pair of stages, the Spear-
man correlation (Rho) of their normalized, averaged gene ex-
pression vectors. We performed hierarchical clustering on these