Transcriptome screen for fast evolving genes by Inter-Specific Selective Hybridization (ISSH)

Experimental design

The ISSH method (Figure 1) confronts in solution complementary transcriptomes of two closely related species with the aim of rescuing transcripts of fast evolving genes. The property of evolving fast implies that such transcripts will disanneal at low stringencies from the heteroduplexes formed by homologous complementary sequences of the two species. Our method was applied to build a cDNA library enriched in fast evolving transcript fragments of brain tissue of the catfish Ancistrus temminckii. We used as the selector species its close relative Ancistrus dolichopterus. To assess the efficiency of the ISSH method we prepared a non-enriched control cDNA library of brain tissue of A. temminckii using standard protocols. The two libraries were sequenced with the FLX Genome Sequencer technology (Roche). We then "blasted" the enriched and control libraries against the complete genome of the zebrafish and analyzed the differences. We also annotated the transcripts producing significant matches and examined their characteristics to highlight the effectiveness of our method. As the zebrafish is not a close relative to our catfish and because the sequences of interest display high sequence divergence, a substantial proportion of the enriched library yielded no significant Blast matches. Therefore, we prepared an EST library of a close catfish relative, Hypostomus gr. plecostomus, belonging to the same subfamily (Loricariidae: Hypostominae), for refining the analyses.

Analysis of sequence divergence

When using the zebrafish genome as reference, the fastest evolving sequences may not find their homologous counterparts due to the distant evolutionary relationship between the zebrafish and our non-model catfish. Thus, performing the same analysis yet using an evolutionary closer reference - our EST database of the catfish Hypostomus gr. plecostomus - may allow a better understanding of the efficiency of ISSH method. The sequence divergence comparisons (Table 1) show again a systematic and significant enrichment in fast evolving sequences in the enriched library as compared to the control library. The difference between the two libraries is generally higher than when using the zebrafish as reference. This is likely explained by the inclusion of a set of faster evolving genes which can now find their homologues in the evolutionary closer Hypostomus reference.

Characteristics of the sequences retained by the ISSH method

In order to better assess the usefulness of the ISSH method we annotated the contigs of the enriched and control libraries according to the information collected from their translated best Blast hit in the Swissprot/Uniprot database with a minimum threshold of E-score ≤ 10e-8. In this way only 60 contigs were characterized in the enriched library (2.5% of all contigs) and 39 in the control library (3.1% of all contigs). The analysis of the annotated sequences will serve to test three predictions that have to be fulfilled if the method achieves its goal. First, as mitochondrial genes evolve significantly faster than the vast majority of nuclear genes they should be more numerous in the enriched library than in the control. The results indicate that mitochondrial genes represent 22.5% versus 8.6% of the annotated contigs of the enriched and control libraries, respectively, fulfilling the prediction. The second expectation concerns the overall correlation between the expression level and gene sequence conservation, where conserved genes are generally expressed at higher rates than fast evolving genes [24]. The expression level of the annotated contigs were approximated by using Unigene database information on the expression level of their orthologous genes in nervous system tissues of the zebrafish or, alternatively, of the mouse or human when the data was not available. Annotated contigs were classified into four categories of gene expression levels (Figure 2). We discarded here the mitochondrial genes which are fast evolving yet possess a high expression level. As expected, the enriched library is essentially composed by genes belonging to the category with the lowest expression-level (58% of the total versus 26% in the control library). The enriched library also shows a depletion of genes in the highly expressed gene categories, which are generally the more conserved ones. The third prediction refers to the observation that genes with tissue-specific expression evolve generally faster than genes with ubiquitous expression, a category in which most housekeeping genes are found [25, 26]. As predicted, our method resulted in an enrichment of non-ubiquitously expressed genes totaling 57% of the annotated contigs of the enriched library versus 35% in the control library. The non-ubiquitously expressed genes are defined as those expressed in less than four tissues according to Unigene expression information.

We emphasize that the sequences of the transcripts annotated using the mRNA refseq database likely represent the most conserved regions of the isolated transcripts dataset, as faster evolving regions will not find their sequence counterparts in the refseq database, which comprises no closely related catfish sequences.

RNA extraction and preparation of the control library

Total RNA was extracted from fresh brain tissue of Ancistrus temminckii (probed species) and its close relative Ancistrus dolichopterus (selector species) using TRIzol reagent (Gibco). We also extracted total RNA from our catfish outgroup reference Hypostomus gr. plecostomus. After quantification and quality verification of the total RNA, mRNA was isolated using the mRNA Isolation Kit (Roche Diagnostics). The SuperScript double-stranded cDNA synthesis kit (Invitrogen) was used to prepare the brain control library of Ancistrus temminckii and also the outgroup reference Hypostomus gr. plecostomus, starting with 1 μg of brain mRNA and the option of oligo(dT) anchor priming for the first strand synthesis step.

The ISSH protocol

The selector pool of mRNA, extracted in this work from Ancistrus dolichopterus, is biotinylated to allow subsequent separation by magnetic particles coated with streptavidin. Biotinylation of 5 μg mRNA was done using the BIO-ULS labeling kit (Kreatech); the final volume was reduce to 7 μl using a Speedvac concentrator. The probed pool of mRNAs extracted from the species of interest Ancistrus temminckii is reverse-transcribed into ss cDNA using a short-tailed random hexamer primer (5'-AGGA-(N)6-3'). We used 1 μg of mRNA (one fifth of the selector's mRNA amount) and 200 ng of the short-tailed random primer in a total volume of 12 μl. The reverse transcription was performed using the SuperScript II RT (Invitrogen) following the manufacturer's protocol for random priming; the final volume was 20 μl. The RNA template is destroyed by alkaline hydrolysis (0.35 N NaOH; 0.35 M EDTA) at 65°C for 15 min. The solution is then neutralized with 0.35 N HCl and first strand cDNAs are purified using the Mini Elute PCR Purification Kit (Qiagen) following the manufacturer's protocol but with an additional washing step and two rounds of elution. The final volume was reduced to 7 μl using a Speedvac concentrator.

Inter-Specific Selective Hybridization

The pool of biotinylated selector mRNA (7 μl) and the pool of first strand cDNA of the species of interest (7 μl) are mixed and the total volume is adjusted to 15 μl. An equal volume (15 μl) of 2× hybridization buffer is added (10 mM EDTA pH8, 1.5 M NaCl, 40 mM sodium phosphate buffer pH 7.2, 10× Denhardt's, 0.2% SDS). The solution is heated at 90°C for 2 min and quickly placed in a rotary shaker located inside a preheated hybridization oven at 55°C. The hybridization is carried on during 60 hours at 55°C. At the end of the hybridization step, 75 μl of NaCl 1 M is added to the hybridization mixture, which is kept at RT.

Separation of the fraction enriched in fast evolving cDNAs

The selector-probed hybridization mix is sequentially denatured to separate two fractions of cDNAs with increasing denaturation stringencies, the first fraction containing the non-hybridized or non-specifically hybridized probed cDNAs and the second fraction is the one enriched in fast evolving transcripts. First, streptavidin magnetic particles (Roche Diagnostics) are prepared according to the manufacturer's instructions (1200 μg) and resuspended in 100 μl of TEN 1000 buffer. The hybridization mixture is then transferred in to the tube containing the streptavidin magnetic particles and placed in a rotary shaker for 45 min at RT. In this step the biotinylated selector mRNAs, which may be hybridized or not with a complementary probed ss cDNA, are linked to the streptavidin magnetic particles. The non-hybridized probed cDNAs are discarded by placing the tube in a magnetic separator (Qiagen) and by removing the supernatant. The magnetic particles with their attached molecules are washed three times at 55°C for 15 min, in 600 μl of preheated 5× SSC, then resuspended in 50 μl of 0.1× SSC and incubated at 65°C for 15 min. In this last step the fast evolving probed cDNAs will disanneal from their selector counterpart and this fraction of interest is recovered in the supernatant after a magnetic separation. This step is repeated once. The fraction enriched in fast evolving cDNAs is purified by ethanol precipitation in presence of ammonium acetate and glycogen. The pellet is rinsed once in 70% ethanol and resuspended in 20 μl water.

Second strand synthesis and adapter ligation

The ss cDNAs are transformed into double stranded (ds) cDNAs using short-tailed random hexamer primers (CCAC-(N)6) and the DNA polymerase I Klenow fragment (Promega), according to the manufacturer's random priming protocol. cDNAs are then blunt ended using T4 DNA Polymerase (Promega), extracted with phenol/chloroform/isoamylalcohol (25:24:1) and recovered by ethanol precipitation with ammonium acetate. Double strand EcoRI adapters (Invitrogen) are ligated to the ds cDNA ends according to the manufacturer's instructions. The final volume is adjusted to 100 μl with water and the cDNAs are purified using the High Pure PCR Product Purification Kit (Roche Diagnostics).

PCR amplification of the fraction enriched in fast evolving cDNAs

A first PCR amplification is performed using a single primer (5'-GTCGACGCGGCCGCGAATT-3') targeted toward the EcoRI adapter ligated at both ends. The PCR reaction is done in 50 μl final volume with 10 μl of cDNA as template and with the following profile: 1 min initial denaturation at 94°C followed by 35 cycles with 30 s at 94°C, 30 s at 62°C, 2.5 min at 72°C and a final elongation step of 5 min at 72°C. The PCR product is checked on 1,5% agarose gel. A nested PCR is performed using specific primers overlapping the end of the EcoRI adapters and the tails of the two short-tailed random primers used for the synthesis of the first strand and then for the synthesis of the second strand (EcoRI-AGGA: 5'-TCGCGGCCGCGTCGACAGGA-3'; EcoRI-CCAC: 5'-TCGCGGCCGCGTCGACCCAC-3'). The PCR conditions are as described above but the amount of template DNA is adjusted according to the result of the first PCR. The PCR products are checked on 1,5% agarose gel and then purified using the High Pure PCR Product Purification Kit (Roche Diagnostics).

High-throughput sequencing

For the Ancistrus control and the outgroup reference Hypostomus gr. plecostomus, shotgun DNA libraries were prepared with a starting amount of 4 μg DNA. The mean fragment size was of about 500 bp, obtained using nebulizers and chemicals from the GS DNA Library Preparation Kit (Roche Diagnostics) according to the manufacturer's manual. This step was not needed for the Ancistrus library enriched in fast evolving transcripts as the ISSH method results in a PCR product containing fragmented transcripts, generally in the range of 300 to 1000 bp. After DNA purification, the DNA end repair step and the ligation of the barcoding adaptors were performed following established protocols [31]. The adapter-ligated DNA from each of the three libraries were pooled and prepared for the 454 sequencing according to standard protocols [32], using the GS DNA Library Preparation Kit with Titanium reagents, and following the instructions of the GS FLX manuals (Roche Diagnostics). The library was sequenced on one 16th region of a full GS FLX sequencing plate with a prior titration run. Upon completion, sequences were screened for primer concatemers, week signal, poly A/T sequences, and barcodes for assigning them to one of the three samples. The average lengths of the reads were 180 bp. cDNA assemblies were performed with the SeqMan software from DNAStar. The cDNA library enriched in fast evolving genes and the control library of Ancitrus temminkii were deposited in the Short Read Archive (SRA) of NCBI under the accession number SRA009346.1

Blast search and transcript annotation

The Blast search against the zebrafish sequences of all the EMBL sub-divisions (Expressed Sequence Tag; High Throughput cDNA sequencing; High Throughput Genome sequencing; mRNA of Standard; Whole Genome Shotgun) were performed on the Vital-IT high-performance computing facility of the Swiss Institute of Bioinformatics http://www.vital-it.ch. We used blast parameter values suitable for comparing divergent sequences (word size = 7; match score = +1; mismatch score = -1; initial penalty for opening a gap = 1; penalty for extending a gap = 2). The local Blast search against our Hypostomus gr. plecostomus brain EST database was performed using the software blast-2.2.19 developed by NCBI. Perl scripts for parsing the blast outputs were built using Eclipse SDK 3.4.1.

The proportion of contigs with low complexity sequences or sequence repeats was assessed using RepeatMasker open-3.2.8 (Smit, AFA, Hubley, R & Green, P. RepeatMasker Open-3.0. 1996-2004; http://www.repeatmasker.org). Transcripts were annotated according to their best Blast hit against Swissprot/Uniprot databases, with a minimal E-score of 10e-8. The translations into the six frames were performed using BCM Search Launcher [33] and blasting was done with Blastp at NCBI. Expected frequency of stop codons in non-coding sequences was calculated by multiplying the three single nucleotide frequencies taken from the sequence data of the corresponding library, and summing the frequency of the three possible stop codons. Gene transcription levels in specific tissues were taken from the Unigene database and are expressed in number of ESTs of the gene under consideration divided by the total ESTs of the tissue library, multiplied by 10'000. Gene ontology classification was performed on Panther (Protein ANalysis THrough Evolutionary Relationships; http://www.pantherdb.org), complemented with ontology information given in Uniprot database. We used only the top categories of the classification hierarchy, as given in Panther. Fast evolving transcripts found in Ancistrus temminckii, Hypostomus gr. plecostomus EST, and in the mRNA reference sequences database of NCBI, restricted to the Teleostei (Blastn threshold E-score < 10e-8), were used to asses the sequence divergence. A tentative annotated was given according to the best hit against the mRNA refseq database. For direct comparison purposes, sequence divergence was calculated on the sequence region present in all three taxa. Sequences of the reference fast evolving markers were obtained from GeneBank: Ancistrus brevipinnis COI: EU359402; Hypostomus boulengeri COI: EU359422; Danio rerio complete genome: NC_002333; Ancistrus cirrhosus RTN4 introns: EU817562; Hypostomus boulengeri RTN4 introns: EU817560. The RTN4 introns from Danio rerio were retrieved from Ensembl http://www.ensembl.org/, locus: chromosome: Zv8:1:42092991:42094205:1.

Data access

Raw read data is available at the Short Read Archive (SRA) of NCBI under the accession number SRA009346.1

No ethical approval was required for this study.