Keywords
RNA-seq, transcriptome, open reading frame, extensin, proline-rich proteins, dehydrins, senescence-associated proteins, Computational genomics, Juglans nigra, black walnut, heartwood/sapwood transition zone
This article is included in the Agriculture, Food and Nutrition gateway.
RNA-seq, transcriptome, open reading frame, extensin, proline-rich proteins, dehydrins, senescence-associated proteins, Computational genomics, Juglans nigra, black walnut, heartwood/sapwood transition zone
In this version, we have
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See the authors' detailed response to the review by Varodom Charoensawan
Analysis of the complete set of RNA molecules in a cell, the transcriptome, is critical to understanding the functional aspects of the genome of an organism. Most transcripts get translated into proteins by the ribosome1. Non-translated transcripts (noncoding RNAs) may be alternatively spliced and/or broken into smaller RNAs, the importance of which have only recently been recognized2. Transcriptional levels vary significantly based on environmental cues3, and/or disease4. Quantifying transcriptional levels constitutes an important methodology in current biological research. Traditional methods like RNA:DNA hybridization5 and short sequence-based approaches6 have been supplanted recently by a high-throughput DNA sequencing method - RNA-seq7,8. Concomitant with the introduction of RNA-seq has been the development of a diverse set of computational methods for analyzing the resultant data9–21.
In the current work, we present a methodology for analyzing RNA-seq data that has been assembled into transcripts (YeATS - Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). The process of associating genomic open reading frames (ORF) to a set of transcripts (transcriptome) is the key step in YeATS, enabling identification and correction of specific errors arising from sequencing and/or assembly, a novel feature missing in most known tools. These errors include transcripts that have not been merged, a transcript having broken ORFs and transcripts containing long repeats. Also, YeATS identifies noncoding RNAs by comparison to compiled databases22, transcripts with multiple coding sequences and highly transcribed genes (based on simple normalization of raw counts followed by sorting).
Here, the YeATS workflow is demonstrated using a representative sample of the transcriptome from the tissue at the heartwood/sapwood transition zone in black walnut (Juglans nigra L.). We have identified transcripts that have sequencing and/or assembly errors (~5%). A novel feature that emerged from our analysis was the presence of a highly transcribed gene that had no known homologous counterpart in the entire BLAST database. The amino acid composition of the longest open reading frame of this gene consists of a high percentage of leucine, histidine and valine, and classifies this as a putative extensin23. Given the economic and ecological importance of black walnut timber, characterization of such genes will enhance our understanding of the mechanisms underlying the unique properties associated with the wood of these trees24. The significance of proline-rich proteins25, dehydrins26, senescence-associated proteins27 and DNAJ28 proteins to the formation of heartwood was established through their transcriptional abundance. Finally, based on transcripts that have no known homologs, we have identified noncoding RNAs by comparison with the noncoding RNA database for Arabidopsis22. Thus, in the current work, we present a workflow (YeATS) with several novel features absent in most currently available software.
The input to YeATS is a set of post assembly transcripts as a fasta file (φTRS). The first step is to identify the set of genes (proteins) encoded by φTRS. This is done by associating a proper open reading frame (ORF) to each transcript. This involves a comprehensive automated BLAST run29.
For each transcript in φTRS, we generate the three longest ORFs (using the ‘getorf’ utility in the EMBOSS suite30) (Figure 1). These three ORFs are BLAST’ed to the full non-redundant protein sequences (‘nr’) database. For a given E-value cutoff (1E-12 in the current work), we create four sets
1. Only one ORF is less than the cutoff - the transcript is uniquely annotated.
2. None of the ORFs is less than the cutoff - the transcript has no known homologs.
3. More than one ORF is less than the cutoff.
(a) The ORFs map to different fragments of the same protein. This points to an error in the sequencing or the assembly, which breaks down the contiguous ORF into two fragments.
(b) The ORFs map to different proteins - these are instances of a transcript having two valid ORFs. We duplicate the transcript, associating each one to a different protein sequence.
To produce the uniquely annotated set of genes, we ignored entries with the keywords chromosome, hypothetical, unnamed, unknown and uncharacterized, in order to have a functional characteristic in the annotation, provided the final annotated entry has low E-value. Also, apart from comparing E-values, we also compare the BLAST score, choosing an ORF as unique if its BLAST score was more than twice any other BLAST score, even if other scores satisfied the E-value criteria.
Algorithm 1 describes the process of merging transcripts (SI Figure 1). For a given length (which varies from 5 to 15 in this case), the 5’ and 3’ sequences and identifiers of each transcript are stored in new string databases: 3’=Begin; 5’=End. Repetitive strings (strings that have only two letters) are ignored, as it is difficult to ensure their uniqueness. For each string of n length in the Begin (3’) string database, we find whether: a) unique matches of n length (one-to-one mapping) are present in the End (5’) string database and b) that the prefixes (initial transcript identifiers) of the transcripts are the same.
Input: φTRS ⇐ Set of transcripts
Output: φTRSMERGED: Pairs of transcripts that can be
merged
begin
φTRSMERGED ← 0;
while NewStatesAdded do
foreach TRSi in φTRS do
φBEGIN ← 0;
φEND ← 0;
foreach len:5..15 do
AddBeginingofTRS(φBEGIN,TRSi,len);
AddEndofTRS(φEND,TRSi,len);
end
foreach stringi in φBEGIN do
/* ignore strings that have less than 3 letters, these are repetitive*/
IgnoreRepeats(stringi);
if(∃ only one stringj in φEND) such that prefixof(TRSi) == prefixof(TRSj))[
φTRSMERGED ←
AddtoMergeableSet(TRSi,TRSj);
]
end
end
end
return φTRSMERGED;
end
Algorithm 2 describes the iterative method for identifying homologous genes in the genome based on the transcriptome. First, the transcriptome is converted to a set of protein sequences by choosing the appropriate ORF (described above) as the representative protein sequence, and a BLAST database (TRSDB) is created. An input protein sequence (possibly from another organism) of a gene of interest is used to query TRSDB using BLAST29. This results in a set of significant transcript matches which is pruned based on a cutoff identity (40% in this case) and the criterion that the sequence length should not differ more than another parameterizable value (50 in this case). Both these transcripts are now potential genes, and the above mentioned process is repeated for each of them, until no new transcripts are added.
Input: G ⇐ Amino acid sequence of gene
Input: TRSDB ⇐ BLAST database of the protein sequences from each transcript, choosing the longest ORF as the representative protein sequence
Input: identitycutoff ⇐ Ignore matches which are less than identitycutoff % identical to the sequence under consideration
Input: lengthcutoff ⇐ Ignore matches where the sequence length differs by more than lengthcutoff % from the sequence under consideration
Output: φgenes
begin
φgenes ← G;
φprocessed ← 0;
NewStatesAdded ← 1;
while NewStatesAdded do
NewStatesAdded ← 0;
foreach Gi in φgenes such that Gi is not in
φprocessed do
φprocessed ← Gi;
= BLAST Gi on TRSDB;
foreach TRSi in do
difflength ←
length(Gi) – length(TRSi) ;
if(identity(TRSi, Gi) > identitycutoff ^
(difflength < lengthcutoff)) [
NewStatesAdded ← 1;
φgenes ← TRSi;
]
end
end
end
/* This is not a TRS, but an input - remove this from the set*/
remove G from φgenes;
return φgenes;
end
The raw counts for each transcript is normalized according to Equation 1, assuming a read length of 100.
The sequence alignment was done using ClustalW31. The alignment images were generated using SeaView32.
The runtimes for most of the processing required in YeATS is a few hours on a simple 16 GB, 16-core machine, barring the search for homologies in the BLAST ‘nr’ database. This search can be significantly accelerated when the organism under investigation has well-annotated protein databases (as in the current case), much in lines of the newly introduced SMARTBLAST (http://blast.ncbi.nlm.nih.gov/smartblast/), to runtimes under a day.
Total RNA was isolated from the xylem region immediately external to the heartwood of a 16 year-old black walnut. The tree was felled in November, cross sections about 1 inch thick were taken from the base and dropped immediately into liquid nitrogen. After the sections were fully frozen they were transported to the lab on dry ice. The transition zone was then chiseled and the xylem was ground using a freezer mill. The RNA was extracted from 100g of ground wood using lithium chloride extraction buffer, and subsequently treated with DNAse (to remove genomic DNA) using an RNA/DNA Mini Kit (Qiagen, Valencia, CA) per the manufacturers protocol. Presence of RNA was confirmed by running an aliquot on an Experion Automated Electrophoresis System (Bio-Rad Laboratories, Hercules, CA).
The cDNA libraries were constructed following the Illumina mRNA-sequencing sample preparation protocol (Illumina Inc., San Diego, CA). Final elution was performed with 16 μL RNase-free water. Each library was run as an independent lane on a Genome Analyzer II (Illumina, San Diego, CA) to generate paired-end sequences of 85bp in length from each cDNA library.
Prior to assembly, all reads underwent quality control for paired-end reads and trimming using Sickle33. The minimum read length was 45bp with a minimum Sanger quality score of 35. The quality controlled reads of 19 libraries from J. nigra were de novo assembled with Trinity v2.0.614 (standard parameters with minimum contig length of 300bp) (manuscript in submission, bioproject id PRJNA232394). Subsequently, the reads from the TZ from J. nigra was aligned to this transcriptome and counts obtained by BWA’s short read aligner v.0.6.2 (‘bwa aln’) (http://bio-bwa.sourceforge.net/)34. The Illumina reads for the transition wood transcriptome can be accessed at http://www.ncbi.nlm.nih.gov/sra/SRX404331.
The input dataset to the YeATS tool was a set of transcripts, transcript identifiers and their corresponding raw counts (see Supporting information), obtained from the tissue at the heartwood/sapwood transition zone (TZ) in black walnut (Juglans nigra L.) (Figure 2). These raw counts were normalized (see Methods), and transcripts with zero counts were ignored (see rawcounts.normalized.TZ in Dataset 1). There were ~24K such transcripts
In order to associate a transcript to a specific open reading frame (ORF), the ORFs of is obtained using ‘getorf’ from the Emboss suite30 (see ORFS.tgz in Supporting information) (Figure 1). The three longest ORFs for each transcript is BLAST’ed to the full non-redundant protein sequences (‘nr’) database, and the results were used to characterize the genes.
There were ~1200 transcripts that had possible sequencing or assembly errors, ~22K transcripts that had significant matches (E-value<E-12) in the ‘nr’ database, 113 transcripts that had lower matches (E-12<E-value<E-08) in the ‘nr’ database, ~700 transcripts that had no matches in the ‘nr’ database and about 200 transcripts that could be merged based on overlapping amino acid sequences. We describe these in detail below.
We observed transcripts that had multiple ORFs that matched to the same gene with high significance (E-value<E-10). The possibility that such an occurrence is not an experimental artifact is low. Transcript C15259_G1_I1 is one such example, having two ORFs - ORF_36 (length = 144) and ORF_9 (length = 122), both of which match to the mitochondrial ATP-dependent Clp protease proteolytic subunit 235 (GenBank: CAN64666.1) from Vitis vinifera with E-values of 6E-92 and 7E-45, respectively. Figure 3 shows the alignment of these two ORFs to the Vitis vinifera protein indicated the possible site of the sequencing error or transcript misassembly. This aspect of the YeATS methodology can be used to estimate the sequencing and transcript assembly error rate. For example, in the current transcriptome of the walnut TZ, we found a 5% (1200 out of 24,000) error rate.
A small number of transcripts had long repeats (on the reverse strand), as identified by transcripts that had multiple identical ORFs. For example, transcript C50369_G5_I2 has two ORFs (length = 143) that matched to an uncharacterized protein (Uniprot id: XP_009362671, E-value= 4e-13). These ORFs were located on the reverse strand, and were exactly the same (Figure 4). There were only 8 such cases.
About ~200 transcripts have been merged using conservative metrics by YeATS (see Methods, list.merge in Supporting information). For example, transcripts C55368_G1_I3 and C55368_G2_I1 were merged based on a stretch of 12 amino acids (NFDENRGALNSH) (Figure 5). The indicated single nucleotide difference might be the reason for the failure of the assembly program to merge these two transcripts. Transcript C55368_G1_I3 had two exact repeats of this stretch, which is a likely assembly error.
Some transcripts were associated with multiple ORFs with distinct significant matches in the ‘nr’ database. We demonstrate this for the transcript C8909_G1_I1, which had two ORFs - ORF_104 (length = 331) and ORF_45 (length = 390) which matched to a clathrin light chain36 (Uniprot id:XP_006481016.1, E-value=3E-126) and a leucine repeat rich receptor-like serine/threonine protein kinase37 (Uniprot id: XP_007026739.1, E-value=0), respectively. These ORFs were on opposite strands, and did not overlap. It was not possible to ascertain which was the correct gene product, and it is a distinct possibility that both strands were transcribed38. A slightly different situation arose when both the ORFs were on the same strand39, as in the case of the transcript C54995_G6_I2. For example, in transcript C54995_G6_I2, there were two ORFs - ORF_157 (length = 464) and ORF_231 (length = 543) that matched to a RING/U-box superfamily protein40 (Uniprot id: XP_007042454.1, E-value=7E-149) and a homeodomain-like superfamily protein isoform41 (Uniprot id: XP_007030696.1, E-value=0), respectively. Both of these proteins were on the same (reverse) strand of the transcript. These transcripts are candidates for chimeric42 or fusion43 genes, since the ribosome is known to bypass small nucleotide stretches separating two ORFs44.
Table 1 shows the transcripts with the highest counts. Interestingly, the most abundant transcript had no homologous counterpart in the full BLAST ‘nr’ or ‘nt’ database (GenBank accession: C52369_G2_I1). A proline-rich protein (PRP), a part of the protein superfamily of cell wall proteins consisting of extensins and nodulins, was found to have the second most abundant transcript23,45. Proline comprises 19% of the amino acids in the ORF of this transcript. PRPs are found as structural proteins in wood, and it was hypothesized that these proteins occur in the xylem cell walls during ligniflication, and influence the properties of wood46. PRPs were associated with carrot storage root formation47, were wound and auxin inducible47 and implicated in cell elongation48. PRPs are also an integral component of saliva responsible for the precipitation of antinutritive and toxic polyphenols by forming complexes49. Two DNAJ/HSP40 chaperone proteins, which are involved in proper protein folding, transport and stress response, showed high transcriptional levels28. Two DNAJ/HSP40 chaperone homologs (GenBank accession id: BI677935 and BI642398) were shown to be differentially expressed during summer at the sapwood/heartwood TZ of black locust50. The transcription levels of dehydrin-related proteins were shown to be seasonally regulated in the wood of deciduous trees26,51. However, this dehydrin protein is homologous to a 24kDa dehydrin (Uniprot id: AGC51777) from Jatropha manihot, a drought resistant plant52, unlike the ~100kDa proteins investigated in 26. Senescence-associated proteins, and the related tetraspanins, were also highly transcribed27. One highly expressed transcript was homologous to a protein that is yet to be characterized.
We demonstrated the (iterative) gene finding methodology in YeATS on a transcription factor that has an AP2 DNA binding motif (RAP2.6L in Arabidopsis, At5g13330)53. This protein showed differential tissue specific expression, and is likely to be involved in plant developmental processes and stress response54. Recently, the sequence of a homolog of RAP2.6L was deduced (Uniprot id: C1KH72, JnRap2) from an EST sequence isolated from tissue at the heartwood/sapwood TZ in black walnut (Juglans nigra L.), and its role in the integration of ethylene and jasmonate signals in the xylem and other tissues was established55,56. Using the sequence of JnRap2, we probed for other RAP2 genes in the TZ of walnut. We found three possible genes (C38523_G2_I1, C53728_G7_I1 and C53728_G7_I2) (Figure 7). It was observed that C53728_G7_I2 was closest to the JnRap2 gene (97.4% identity, 98.2% similar), and is probably the same gene. C53728_G2_I1 was also significantly homologous to the JnRap2 gene (84.4% identity, 92.4% similar), and it appears to be an allelic or splice variant, a conflict that can be resolved after the publication of the complete walnut genome. Raw counts (see Supporting information) demonstrated that the transcript C38523_G2_I1 had negligible expression levels in TZ, corroborating the previous detection of only one RAP2 protein in 55.
The top three ORFs of ~600 transcripts had no match in the BLAST ‘nr’ database. Although these may be unique genes, another possibility that must be considered is that these are non-coding RNA genes2. The nucleotide sequences of these 600 transcripts were BLAST’ed to the database of noncoding RNAs in Arabidopsis22. Three matches were identified: C52424_G5_I11, C52424_G5_I4 and C53565_G3_I1. Both C52424_G5_I11 and C52424_G5_I4 are homologous to CR20, a cytokinin-repressed gene in excised cotyledons of cucumber, hypothesized to be non-coding RNA57. Analogous to the current work, the CR20 gene had alternate splicing57. C53565_G3_I1 had a 100% match to the Arabidopsis locus ATMG01380, a mitochondrial 5S ribosomal RNA, which is a component of the 50S large subunit of mitochondrial ribosome58.
High-throughput mRNA sequencing (RNA-Seq) has revolutionized the field of transcript discovery, providing several advantages over traditional methods7,8. Following isolation and fragmentation of RNA and subsequent generation of cDNA libraries, a high-throughput sequencing platform is selected to generate short reads59. Reconstruction of transcripts from these short reads (assembly) may be performed using a reference genome or de novo algorithms15–18,21,60. Sequencing biases, variable coverage, sequencing errors, alternate splicing and repeat sequences are some of the challenges faced by these assemblers14,61.
Several post assembly computational tools provide further curation of transcripts resulting from the assemblers. The curation step involves identifying redundancies19,20, finding coding regions62, annotating the transcripts (https://transdecoder.github.io/) and detecting inaccuracies by aligning the transcripts to the genome63. In the current work, we present an integrated workflow for RNA-seq analysis (YeATS). YeATS includes most features of the tools mentioned above. Additionally, YeATS delivers several capabilities absent in these tools. A comprehensive BLAST analysis of the top three open reading frames of each transcript enables the identification of erroneous transcripts arising out of sequencing or assembly errors. These erroneous transcripts can be classified as: a) transcripts that have not been merged, b) transcripts that result in broken ORFs and c) transcripts that have long improbable repeats. Finally, YeATS provides annotation of the genes, enumerates homologous genes based on a template sequence and specified similarity threshold and identifies transcripts with multiple ORFs. The ribosome is known to bypass small nucleotide stretches separating two ORFs44. These are rare events, however, and thus unlikely to apply to the ~1200 transcripts that have broken ORFs pointing to the same gene64. Transcripts having multiple ORFs on the same strand are good candidates for chimeric42 or fusion43 genes dependent on ribosome bypassing.
The current work reveals and corroborates several aspects of the biology of hardwood trees. Probably, the most interesting is the detection of a highly transcribed gene (C52369_G2_I1) with no known homologs in the complete protein and nucleotide BLAST database, or significant matches in a database of long non-coding RNA genes22. If indeed the longest ORF of this transcript encodes a protein, it is 143 amino acids long, and is leucine (18%), histidine (13%) and valine (10%) rich (Figure 8). Although it is likely that this is a protein with leucine rich repeats, these proteins are typically larger proteins65. On the other hand, histidine and valine rich extensins have been reported to be constituents of plant cell walls of dicots23. The regulatory stimuli of extensins are different for monocots (which also have different amino acid composition) and dicots23. A significant presence of extensin-like proteins in the cell wall of both developing and mature xylem (wood) have been reported for pine46,66. The publication of the walnut genome will aid the characterization of these genes by elucidating its promoter sequences.
Well characterized proteins like proline-rich proteins25,46, dehydrins26, senescence-associated proteins27 and DNAJ/HSP40 chaperone50 proteins were also abundant in the transcriptome. While Arabidopsis supports secondary growth, it fails to accumulate wood; it is therefore interesting to identify highly transcribed genes that are missing in the Arabidopsis proteome (Table 2). The DNAJ/HSP40 chaperone, dehydrins and tetraspanin proteins are found in the Arabidopsis proteome (TAIR10_pep_2010121467), while the putative extensin, the proline-rich protein, a probable zinc transporter protein, an uncharacterized protein and senescence-associated protein appear to be unique to the walnut proteome.
Also, we corroborated the presence of a transcription factor that has a AP2 DNA binding motif53,55, and identify additional splice/allelic variants with similar transcriptional levels. Once again, the knowledge of the walnut genome would enable a more profound understanding of such genes.
In summary, the current work elucidates an integrated workflow for RNA-seq analysis with several innovative features for identifying and correcting erroneously assembled transcripts. We demonstrated this workflow by characterizing the transcriptome of the tissue at the heartwood /sapwood TZ in black walnut.
F1000Research: Dataset 1. YeATS Dataset, 10.5256/f1000research.6617.d4973068
The YeATS tool suite was designed by Chakraborty, Britton and Wegrzyn did the analysis of the transcriptome, Woeste isolated the RNA, Butterfield was involved in the validation. The rest of the authors were involved in various aspects of the study design. Chakraborty wrote the first draft and the rest of the authors were involved in the editing.
The authors wish to acknowledge support from the California Walnut Board and UC Discovery program.
I confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
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