Abstract
Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the causal mutation by traditional mapping and candidate gene sequencing is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in Caenorhabditis elegans mutants. First, we report our use of restriction site-associated DNA (RAD) polymorphism markers for rapidly mapping mutations after chemical mutagenesis and mutant isolation. Second, we describe our use of genomic interval pull-down sequencing (GIPS) to selectively capture and sequence megabase-sized portions of a mutant genome. Together, these two methods provide a rapid and cost-effective approach for positional cloning of C. elegans mutant loci, and are also applicable to other genetic model systems.
DETERMINING mutant gene identity is a key step for understanding gene function in forward genetic screens following mutagenesis and phenotype-based mutant isolation. In some organisms such as fungi and bacteria, a recessive mutant allele can be complemented with a plasmid-borne wild-type gene to establish gene identification. In organisms that lack robust DNA transformation methods, mapping with visible or selected single nucleotide polymorphism (SNP) markers to progressively finer genomic intervals is the traditional route to ascertain identity of the mutant gene. Now whole genome sequencing (WGS) methods can significantly reduce the time required to identify the causal mutation. For example, WGS can simply be used to determine all of the sequence alterations present in a mutant strain (Sarin et al. 2008; Smith et al. 2008; Srivatsan et al. 2008; Blumenstiel et al. 2009; Irvine et al. 2009). However, some mapping data are still required to differentiate the background mutational load from the causal mutation. More recently, WGS has been performed on outcrossed mutant progeny to combine mapping and sequencing for pinpointing the position of the causal mutation (Doitsidou et al. 2010; Zuryn et al. 2010).
While resequencing a genome to identify mutant alleles is being used more frequently, in some cases it is more efficient to sequence only a portion of a genome. For example, sequencing of a single chromosome, a defined genomic interval, exonic sequences, or a single locus can be more cost effective when there is evidence that a mutation resides within a specific genome feature. There have been several throughput-enhancing advances in capturing targeted regions of a genome using DNA annealing since the first reported use of this methodology whereby individual microarray spots were physically scraped from the substrate (Ksiazek et al. 2003; Rota et al. 2003; Wang et al. 2003). For example, genomic DNA can be annealed to microarrays printed with oligonucleotides covering the region to be targeted, washed, and then eluted for sequencing (Albert et al. 2007; Hodges et al. 2007; Okou et al. 2007). Alternatively, oligonucleotides can be used to capture homologous genomic DNA in solution (Gnirke et al. 2009). While these approaches are extremely high throughput, they also can be prohibitively expensive.
We have developed two Illumina-based sequencing methods in Caenorhabditis elegans that offer an alternative pipeline for mutation detection. First, we have performed restriction site associated DNA (RAD) polymorphism mapping to position the causal mutation to a relatively small region of the genome. Second, we have used genome interval pull-down sequencing (GIPS) to sequence a defined genomic interval. Genome intervals are captured by annealing sheared genomic DNA to sheared fosmids containing wild-type C. elegans DNA, eliminating the need for customized microarray or oligonucleotide production. Because multiple RAD mapping and genome interval sequencing samples can be combined in a single Illumina lane, it is possible to positionally clone and identify the mutant loci rapidly and cost effectively without performing WGS.
Materials and Methods
C. elegans strains and culture
Strains were grown under standard laboratory conditions (Brenner 1974). The temperature-sensitive (ts) mutants were maintained in a 15° incubator and shifted to a 26° incubator to perform temperature upshifts for determining embryonic lethality. Mutants were isolated in a lin-2(e1309) background, as previously described (Encalada et al. 2000).
Genetic crosses for RAD mapping
To map or1167ts, we crossed the polymorphic C. elegans strain CB4856 into the original mutagenized background [or1167ts/or1167ts; lin-2(e1309)/lin-2(e1309)]. After self-fertilization of the heterozygous F1 outcross, we pooled 200 of the 1/16 of the F2 progeny that were again or1167ts/or1167ts; lin-2(e1309)/lin-2(e1309), taking advantage of the lin-2 egg-laying defect to identify with a stereomicroscope within F1 self-progeny or1167ts/or1167ts; lin-2(e1309)/lin-2(e1309) F2’s filling up with dead embryos [avoiding laboriously singling out hundreds of F2’s to look for production of dead embryos by egg-laying lin-2(+/+ or +/e1309) F2 progeny]. Similarly, for mapping unc-13, we also performed a cross to CB4856 but selected ∼200 Unc F2 progeny for the genomic DNA preparations. For mapping or1089ts, we crossed the original mutant to CB4856 males and isolated 800 F2 hermaphrodites that were tested for embryonic lethality. Approximately 200 homozygous or1089ts animals were recovered and used for the RAD mapping procedure.
Illumina sequencing for RAD mapping
Genomic DNA was isolated from pools of ∼200 homozygous unc-13 F2’s, and ∼200 or1054ts; lin-2(e1309) F2’s as well as the N2 and CB4856 parental strains using the Qiagen DNeasy kit. A total of 150 ng of each sample was digested with EcoRI and processed into barcoded RAD libraries as previously described (Baird et al. 2008) with the minor modification of using the paired end P2 adapter (Hohenlohe et al. 2010). Briefly, each sample was individually digested with EcoRI, and a P1 adapter (with a 4-bp barcode; see below) was ligated to the overhangs. After this step multiple samples were multiplexed. Next, the DNA was sheared and gel extracted to obtain ∼400-bp fragments and the Illumina P2 adapter was ligated. Samples were then run on the Illumina flow cell. For RAD mapping, there is no need to use an Illumina kit (for full protocol see Baird et al. 2008). The RAD library from the mutant pool was sequenced at ≥30× coverage in an Illumina Genome Analyzer IIx machine. With SNPs present at about every 1000 bp in the polymorphic CB4856 strain, and sequencing reads of ∼75 bp from each EcoRI site, we anticipated detecting a SNP near 1 in 10 EcoRI sites, or about one every 50,000 bp, which was close to the observed value of one SNP in 64,000 bp achieved, on average. The RAD sequences were aligned to the reference Bristol N2 genome using the Bowtie software package (Langmead et al. 2009). The Bowtie output was then exported to SAMtools (Li et al. 2009) and converted into BAM format. We then produced a pileup file, to which we applied the samtools.pl script “varFilter” command (using default options) to identify SNPs. The varFilter results were then saved as a tab-delimited file for use with graphing software (Microsoft Excel and Adobe Illustrator). As an alternative method for identifying N2/CB4856 SNPs, one could use the MAQGene program (Bigelow et al. 2009), which may be more accessible to nonbioinformaticians (as used by Doitsidou et al. 2010).
Illumina sequencing of genomic intervals
To pull down intervals of genomic DNA to which or195ts, semidominant or600(sd),ts, and or683ts were mapped, we used magnetic bead pull-downs. A total of 5 μg of genomic DNA was purified from each mutant strain using a DNeasy Blood and Tissue kit (Qiagen) and sheared to an average size of 500 bp by sonication in a Bioruptor (Diagenode). The ends of the sheared DNA fragments were blunted using a QuickBlunt kit (New England Biolabs) and the fragments purified with a PCR purification kit (Qiagen). A-overhangs were added to the genomic fragments by incubation of the purified, blunted DNA with 150 units of Klenow DNA polymerase exo- (New England Biolabs) and dATP at 37° for 30 min. The modified fragments were purified with a mini-elute PCR purification kit (Qiagen). A total of 7 μl of 1-μM modified Illumina sequencing adapters were ligated to the sheared genomic fragments at 16° for 2 hr using 2000 units of T4 DNA ligase (New England Biolabs). [Top strand: 5′ ACACTCTTTCCCTACACGACGCTCTTCCGATCxxxx*T 3′; bottom strand: 5′ phosphate xxxxGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 3′ (where x indicates the barcode bases and *, phosphorotioate bond.) The ligation reaction was size separated by agarose gel electrophoresis, and fragments between 150 and 500 bp in size were purified from the gel using a Gel Extraction kit (Qiagen). The purified ligation products were PCR amplified using Phusion high fidelity DNA polymerase (New England Biolabs) and the Illumina amplification primers 5′ AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT 3′ and 5′ CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT 3′. The following cycling conditions were used for PCR: 98° for 2 min, 15 cycles of 98° for 10 sec, 65° for 30 sec, and 72° for 15 sec. Following amplification, samples were size separated by agarose gel electrophoresis, and fragments between 150 and 500 bp were purified with a Gel Extraction kit (Qiagen).
Biotinylated probe preparation:
DNA preps homologous to the targeted genomic interval were prepared from genomic fosmids, using a nearly genome-wide fosmid library for C. elegans that was developed by the Genome Sciences Centre in Vancouver, BC, Canada. A total of 100 ng of the fosmid DNA mixtures were combined with 20 μl of 2.5× random octamer solution (Life Technologies) and heated to 100° for 5 min. The mixture was rapidly cooled in an ice/water bath, following which 5 μl of biotin dNTP mixture [1 mM biotin-14-dCTP, 1 mM dCTP, 2 mM dATP, 2 mM dGTP, and 2 mM dGTP, in 10 mM Tris-HCl (pH 7.5), 1 mM Na2 EDTA] (Life Technologies) was added, along with 1 μl of Klenow fragment DNA polymerase (Life Technologies) and ultrapure water to bring the reaction volume to 50 μl. The reaction was then incubated at 37° for 1 hr, following which, the products were size separated on an agarose gel and the predominant 100-bp product was purified with a Gel Extraction kit (Qiagen).
Streptavidin bead preparation:
A total of 50 μl of M270 streptavidin Dynabeads (Life Technologies) were washed three times with 100 μl of 6× SSC and resuspended in 100 μl of bead block buffer [2% I-Block (Tropix), 0.5% SDS, 1× PBS]. Beads were incubated at room temperature for 30 min with occasional mixing and were then magnetically captured and washed three times with 6× SSC.
Hybridization, immobilization, elution, and sequencing:
A total of 5 μg of adapted, purified genomic DNA was combined with 150 ng of purified biotinylated probe in 300 μl of hybridization buffer [54% formamide, 1× SSC, 1% SDS, 5.4× Denhardt’s solution (Sigma), 1 mg/ml Salmon sperm DNA (Life Technologies)]. The mixture was heated to 100° for 2 min and then transferred to a 42° incubator, where it was incubated with mixing overnight. Following overnight incubation, biotinylated probe/genomic DNA fragment hybrids were immobilized by binding to prepared blocked and washed streptavidin beads by combining the hybridization mixture (300 μl) with the bead/SSC mixture (100 μl), and incubating at room temperature for 15 min with occasional mixing. Beads were then magnetically captured, and washed three times with wash solution 1 (1× SSC, 0.15% SDS), three times with wash solution 2 (0.2× SSC), and three times with wash solution 3 (0.05× SSC). After the final wash step, the beads were resuspended in 200 μl of ultrapure water, heated to 100° for 2 min, and quickly magnetically captured. The supernatant was carefully collected and concentrated to a volume of 20 μl in a Speedvac concentrator (Savant). A total of 10 μl of the concentrated supernatant was then used as template for a PCR reaction utilizing Illumina amplification primers and Phusion high fidelity DNA polymerase (New England Biolabs) (2 min 98°, 24 cycles of 98° for 10 sec, 65° for 30 sec, and 72° for 15 sec). The PCR products were purified with a PCR cleanup kit (Qiagen), quantified, and submitted for Illumina sequencing on an Illumina Genome Analyzer II.
Results
RAD mapping of C. elegans mutations
To rapidly map C. elegans mutations, we have used an Illumina sequencing-based genome-wide single nucleotide polymorphism mapping procedure called RAD polymorphism mapping (Lewis et al. 2007; Miller et al. 2007; Baird et al. 2008). RAD markers are SNPs adjacent to restriction enzyme recognition sequences in the genomes of divergent strains. In our case, we used the N2 background (isolated in Bristol, UK) to isolate mutants and subsequently crossed them to the polymorphic Hawaiian CB4856 strain for mapping. The N2 and CB4856 genome sequences have diverged substantially but their hybrid progeny are fertile. On average, there is a SNP approximately every 1 kb, allowing physical mapping using a large number of markers.
To experimentally identify RAD tags, we crossed wild-type N2 hermaphrodites to CB4856 males. F1 hybrid progeny were isolated and genomic DNA was digested with EcoRI. After ligation of Illumina adapters and selective amplification of the RAD tags (Figure 1), Illumina sequencing was performed with an Illumina Genome Analyzer IIx system. Selective amplification was carried out by using a ‘‘Y’’ adapter for the P2 adapter, which prevents fragments that lack a P1 adapter from being amplified after first round synthesis initiated from the P1 site (as described in Coyne et al. 2004; Baird et al. 2008). We detected 3,462 SNPs with an average distance between them being 29 kb. Most SNPs (95%) were separated from an adjacent SNP by <100 kb (Figure 2). The largest distance separating adjacent SNPs occurred near the center of chromosome V (515 kb). Most of the SNPs we identified could be predicted in silico from the sequence of the CB4856 strain (data not shown). Because the sequencing is done with purified RAD tags instead of total genomic DNA, multiple samples can be multiplexed on a single lane in an Illumina sequencer, with each sample containing unique barcodes for subsequent sequence data deconvolution. The barcodes used for RAD mapping are 6-bp sequences added to the P1 adapter primer. In one test, we used one Illumina Genome Analyzer IIx lane to process 13 RAD mapping crosses. We used single-end sequencing with 80-bp reads, to achieve 50 million reads yielding ∼40× coverage. We tested the applicability of RAD mapping coupled with Illumina sequencing using three different approaches.
Restriction site-associated DNA (RAD) mapping schematic. Genomic DNA was isolated from 200 pooled F2 progeny in crosses between the N2 mutants and the polymorphic CB4856 (Hawaiian) strain. Genomic DNA was digested with EcoRI and P1 Illumina adapters were ligated to the fragments. This DNA was mechanically sheared and Illumina P2 adapters were ligated to the fragment ends. Next, RAD tags were selectively amplified and sequenced from the Illumina sequencing primer site encoded on the P2 adapter on an Illumina Genome Analyzer IIx machine.
EcoRI-associated RAD tag locations. (A) RAD mapping results from an N2/CB4856 cross. Vertical lines represent EcoRI-associated RAD markers on each of the C. elegans chromosomes. The total chromosome sizes are listed on the right. For a list of the RAD markers, sequences, and positions, see File S1. (B) Distance between adjacent RAD markers. The plot represents the number of RAD marker pairs at the indicated distances, in kilobase pairs.
First, we mapped a known mutant, unc-13(e450). We crossed the unc-13 mutant to the polymorphic C. elegans strain CB4856 and pooled 200 F2 progeny that were homozygous for the unc-13 mutation. We chose 200 recombinants as a goal because it afforded a relatively large number of independent recombination events, although it is possible that using fewer recombinant F2’s would also yield sufficient resolution. After producing a RAD library from the F2 genomic DNA sample, we performed sequencing on the Illumina machine to detect SNPs across the genome (see Materials and Methods). We used graphing software (Microsoft Excel and Adobe Illustrator) to plot the ratio of CB4856/Bristol SNPs across the C. elegans genomic sequence, indicating the fraction of samples for any one SNP that correspond to the polymorphic CB4856 sequence (Figure 3A). In this test, we identified 683 RAD tags throughout the genome. The ratio of CB4856 to N2 SNPs was ∼0.5 across the genome, except for chromosome I, where a large trough was present. The center of the trough on chromosome I is within ∼800 kb of the known location of unc-13 (Figure 3B). We conclude that Illumina-based RAD mapping can quickly provide the approximate physical position of mutant loci.
RAD mapping results for unc-13. (A) Genome-wide RAD mapping results for unc-13 after crossing it to CB4856. A total of 683 SNPs across the genome in F2 progeny were detected and the ratio of the CB4856 SNPs plotted along the chromosomes. The vertical axis represents the percentage of CB4856 SNPs in the F2 population. (B) Magnification of chromosome I. The trough on chromosome I correlates with the known location of unc-13.
We similarly applied RAD mapping to or1089ts, a mutant of unknown molecular identity with severe defects in mitotic spindle assembly in one-cell–stage embryos (Figure 4A). In this case, we identified 3134 RAD markers in the F2 RAD library. Chromosome I was highly enriched for N2 DNA (Figure 4B), with the trough of N2 DNA centered at 9.25 Mb on chromosome I (Figure 4C), positioning the or1089ts mutation near the center of chromosome I. In a 1-Mb region centered on the trough, we found one candidate gene in online databases, spd-2, that when reduced in function using RNAi, results in defects that closely resemble the or1089ts mutant phenotype (Sonnichsen et al. 2005; Harris et al. 2010). The center of the reduced CB4856 ratio is 276 kb to the left of the known location of spd-2 (Figure 4C). We Sanger sequenced the spd-2 gene in genomic DNA from or1129ts mutants after amplifying the locus using gene-specific primers. We found a single nucleotide mutation that changes a phenylalanine codon to an isoleucine codon at amino acid 544 (Figure 4D). We also found that or1089ts failed to complement spd-2(or293ts) (data not shown). We conclude that or1089ts is a new spd-2 allele.
Phenotype and RAD mapping of spd-2(or1089ts). (A) Defective mitotic spindle formation and cytokinesis failure in an early or1089ts embryo produced from a worm shifted to 26° for 6 hr (lower) compared to a wild-type embryo (upper). (B) Genome-wide RAD mapping of or1089ts. A reduction of CB4856 DNA on chromosome I results from the selection of the mutant homozygotes. (C) The center of the trough is near 9 Mb and is positioned near the causal mutation (see text). (D) DNA sequence analysis of or1089ts identifies a mutation in spd-2 open reading frame (GenBank: AY340594.1). The mutation, causing a phenylalanine-to-isoleucine change in codon 544, is shown relative to the changes of or183 and or188, two known temperature-sensitive alleles of spd-2.
We have screened for temperature-sensitive, embryonic-lethal C. elegans mutants using an egg laying-defective (Egl) lin-2(e1309) mutant background that enables one to screen mutagenized populations for animals that produce inviable embryos without singling out individual worms (see also Kemphues et al. 1988; Encalada et al. 2000; Jorgensen and Mango 2002; O’Rourke et al. 2011). To take further advantage of this Egl background for RAD mapping, we crossed or1167ts; lin-2(e1309) hermaphrodites to CB4856 males and obtained F2 animals that were mutant for both lin-2(e1309) and or1167ts. Instead of testing embryonic lethality of ∼800 F2 animals individually on plates, as was done for spd-2(or1089ts), we were able to isolate ∼200 or1167ts; lin-2(e1309) animals that accumulated mostly dead embryos from a mixed F2 population after shifting them to the restrictive temperature as L4 larvae. After preparing and sequencing the RAD library, we identified >3400 RAD tags. The ratios of CB4856 to N2 DNA were plotted and we found two troughs that correspond to an enrichment of N2 DNA (Figure 5). The trough on the X chromosome is 550 kb from the known location of lin-2, while the trough on the right arm of chromosome IV presumably correlates with the location of or1167ts. The fact that the lin-2 trough does not reach zero likely relates to inadvertently picking some worms that were in fact not lin-2, as even wild-type worms sometimes hold their embryos and can appear Egl. We found one candidate gene, sas-6, positioned 250 kb left of the center of the chromosome IV trough. Like animals depleted for sas-6, or1167ts animals shifted to the restrictive temperature as L4 larvae produce embryos that appear to assemble monopolar mitotic spindles in early embryonic cells (not shown). We sequenced the sas-6 locus with the Sanger method after amplification of the region by PCR. We found a single missense mutation that changes an aspartic acid to a valine in the ninth amino acid of SAS-6 (Figure 5D). As or1167ts also failed to complement a known sas-6 mutant (not shown), we conclude that or1167ts is a sas-6 allele. This example demonstrates that RAD mapping can be easily applied in the context of the lin-2(e1309) marker to minimize the effort required to isolate F2 animals that are homozygous for embryonic lethal mutations. We continue to explore the use of RAD methodologies to rapidly map temperature-sensitive embryonic lethal C. elegans mutants and note that this approach can be used to map virtually any locus that can be assayed in N2/CB4856 F2 animals.
RAD mapping results for a sas-6(or1167ts); lin-2 double mutant. or1167ts; lin-2 mutants were crossed to CB4856 males and F2 progeny, homozygous for both the or1167ts embryonic lethal mutation and the lin-2 mutation, were isolated. (A) Genome-wide RAD mapping results show enrichment for N2 DNA on chromosomes IV and X. (B) The sas-6 gene is located near the chromosome IV trough. (C) The trough on the X chromosome lies near the known position of the lin-2 locus. (D) The sas-6 locus contains a missense mutation that changes an aspartic acid codon to a valine (NCBI: NP_502660.1). An alignment was performed with the C. elegans (Cel) wild-type SAS-6, C. elegans SAS-6(or1167ts), C. remanei (Cre) SAS-6, and C. briggsae (Cbr) SAS-6.
Illumina-based GIPS
To quickly and more cost-effectively identify causal mutations in mutant strains, we have also applied Illumina DNA sequencing to defined genomic intervals, rather than sequencing entire mutant genomes (as has been done to identify some mutant loci in C. elegans (Sarin et al. 2008; Zuryn et al. 2010). Sequencing an entire genome involves more cost than our procedure because we multiplexed multiple barcoded sequencing experiments on a single Illumina Genome Analyzer IIx flow cell. Briefly, we used wild-type genomic DNA from defined genomic intervals, linked to magnetic beads, to partially purify regions of mutant genomic DNA (see Figure 6A and Materials and Methods). We first tested the feasibility of using interval pull-downs and Illumina sequencing by resequencing a previously identified mutation present in the dhc-1 locus of dhc-1(or195ts) mutants worms (O'Rourke et al. 2007). We then identified the mutations responsible for conditional lethality in two previously reported mutants [tbb-2(or600sd,ts) and plk-1(or683ts); O'Rourke et al. 2011], after sequencing genomic intervals of 1.8 and 1.3 Mb, respectively (Figure 7).
Genome interval pull-down sequencing (GIPS) using the Illumina platform. (A) Schematic overview of the interval pull-down sequencing method. First, fosmids of wild-type DNA covering a region of the genome are purified, sheared, and ligated to biotinylated adapters. Next, mutant genomic DNA is sheared and annealed to the biotinylated fosmids. After purification and release of the mutant DNA using magnetic beads, the fragments are subjected to sequencing on an Illumina machine. Finally, the reads are assembled onto the genome scaffold and polymorphisms are identified. (B) Example output of mutant genome assembly. Shown is a small region of the tbb-2 locus with portions of reads aligned beneath the reference sequence. In this case, each read shows that a cytosine in wild type has been changed to a thymidine in tbb-2(or600sd,ts). Also shown is one apparent sequencing error where an A > G change was called in one of the reads. Nucleotides are color coded, uppercase letters, and periods in the sequence reads represent identity with the reference sequence, while reads containing lowercase letters and commas represent sequence data obtained from the reverse complement strand.
Analysis of the genome interval pull-down sequencing. The protocol shown was applied to three mutants. The positions of nucleotide alterations vs. the reference sequence is shown in graphical form. Each line represents a single change, diagonal lines attached to the top of the vertical lines represent multiple changes located very close together. Lines are color coded to show various types of mutations. The ^ in the black bars point to the causative mutations. (A) Sequencing results for the 15 kb dhc-1(or195ts) locus purified using a single genomic fosmid clone. The previously identified missense mutation was found. (B) Sequencing results for a 1.82-Mb region on chromosome III in the or600sd,ts genome. We identified 45 total mutations in the region including the missense mutation in tbb-2 (see Table 1 for details). (C) Sequencing results for a 1.3-Mb region on chromosome III in the or683ts genome. We identified 29 total mutations in the region including the missense mutation in plk-1 (see Table 2 for details).
To test this methodology, we first selected a fosmid that includes the entire dhc-1 locus, available from Source BioScience (http://lifesciences.sourcebioscience.com). We purified the fosmid, sheared it, and linked the fragments to biotinylated beads as described in Materials and Methods. After using these beads to isolate sheared dhc-1(or195ts) genomic DNA, the mutant genomic DNA was eluted and prepared for Illumina sequencing (see Materials and Methods). We aligned 66,853 30-base reads to the 30.1 kb dhc-1–containing fosmid on chromosome I and the average coverage for each position in the fosmid was 66×. For comparison, we also identified 570,311 reads that could be aligned to the rest of the genome, yielding an average read coverage of 0.17× for each nucleotide position. Therefore, we achieved a 388-fold enrichment for reads in the targeted region using our interval pull-down sequencing method. We identified the previously sequenced C-to-T dhc-1(or195ts) mutation in a total of 66 reads, and no other mutations were detected in the dhc-1 locus (Figure 7A). We conclude that GIPS can readily identify the mutations present in relatively large regions of the genome.
Next, we used GIPS to identify the mutation responsible for the early embryonic cell division defects caused by the semidominant, temperature-sensitive mutation or600sd,ts. We defined the or600sd,ts interval using standard mapping crosses with visible morphological and behavioral markers. We localized the mutation to chromosome III between positions 3,618,381 and 5,447,436. We used the WormBase genome browser to identify a minimal tiling path using genomic DNA from fosmid clones available from Source BioScience (http://lifesciences.sourcebioscience.com). We identified 65 fosmids that spanned the region with 7 gaps that totaled <45 kb (∼2.5% of the region). We purified, sheared, and linked the fosmid fragments to biotinylated beads. After using these beads to isolate sheared or600sd,ts genomic DNA, the mutant genomic DNA was eluted and prepared for Illumina sequencing (see Materials and Methods). We found 1,596,403 48-base reads that could be aligned to the region, giving an average coverage for each nucleotide in the interval of 42×. The total number of reads corresponding to the C. elegans genome was 6,034,221.
We used software (SAMtools) to output a text file that lists the mutations identified in the interval. One can also view the sequence reads aligned to the reference wild-type genome sequence with SAMtools (Figure 6B). There were 45 mutations in the 1.8-Mb or600sd,ts interval (Figure 7B). We found 14 extragenic changes, 23 intronic changes, one mutation in a pseudogene, two mutations that cause the transcript to go out of frame, three potential annotation errors, and two missense mutations (Table 1). As >90% of our identified temperature-sensitive mutations are caused by missense mutations (the remaining are due to premature stop codons, small deletions, and mutations in splice-site boundaries) (O'Rourke et al. 2011), we narrowed our analysis of mutations in exon and intron splice sites. Three mutations that appeared to cause exonic changes are likely not the causal mutation because they were present in wild-type DNA; expressed sequence tags show the same mutations, perhaps indicating errors in the reference sequence (we have labeled these as “annotation error?” in Table 1). None of the intronic changes occurred at intron boundaries and thus are unlikely to interfere with RNA splicing. The four remaining exonic mutations occur in the tbb-2, ras-2, his-70, and clec-154 genes. Single nucleotide deletion and insertion mutations in the his-70 and clec-154 loci, respectively, encode proteins with altered C termini, while the ras-2 and tbb-2 mutations are missense. Of these four genes, only RNAi that targets the tbb-2 locus phenocopies the or600sd,ts early embryonic phenotype [note that tbb-2(RNAi) also depletes the paralogous redundant tbb-1 gene product]. Depletion of the other three genes by RNAi does not result in any lethal phenotypes (WormBase). On the basis of sequence data, the embryonic phenotype and genetic interactions with a previously isolated tbb-2 allele, or362sd,ts (O'Rourke et al. 2011), we conclude that or600sd,ts is a tbb-2 allele.
We also identified a recessive temperature-sensitive embryonic lethal mutant, or683ts, that mapped between 6,862,157 and 8,214,712 on chromosome III, on the basis of mapping crosses with visible markers and some PCR-based SNP mapping (not shown). To identify all of the mutations in the interval, we again performed GIPS, as detailed for dhc-1(or195ts) and tbb-2(or600sd,ts). There were 197,752 48-base reads (from a total of 5,616,109 reads) that aligned to the region, giving an average coverage of 7× for the interval. Even though the sequencing coverage was greatly reduced compared to the dhc-1(or195ts) and or600sd,ts results, we were still able to identify 29 mutations in the interval after performing Illumina sequencing (Figure 7C). We found nine intergenic changes, eight mutations in introns, two mutations in 3′-untranslated regions, one mutation in a transposon, six insertions or deletions that caused frame shifting, two potential annotation errors, and one missense allele (Table 2). The annotation errors may represent the wild-type sequence because expressed sequence tags show this same alteration. The single missense mutation changes a methionine codon to a lysine codon in the plk-1 gene. Since or683ts contains a mutation in the plk-1 gene and fails to complement a known allele of plk-1 (O'Rourke et al. 2011), we conclude that or683ts is a novel plk-1 allele.
Discussion
The utility of C. elegans as an animal model, in which one can readily isolate temperature-sensitive mutations in essential genes, and the power of next generation DNA sequencing for greatly reducing the time required to positionally clone mutant loci, now make it possible to much more rapidly isolate experimentally useful conditional mutations in essential genes. Our two new Illumina-based sequencing methods should allow for increased throughput when analyzing large numbers of mutants.
RAD polymorphism mapping has been used successfully to map genes in Drosophila (Miller et al. 2007), threespine stickleback fish (Miller et al. 2007), Neurospora (Lewis et al. 2007), diamondback moths (Baxter et al. 2011), barley (Chutimanitsakun et al. 2011) and now C. elegans. So long as a hybrid strain is available to generate F2 progeny, the methodology should be feasible in any organism. RAD mapping makes it possible to simultaneously and rapidly determine the approximate location of large numbers of mutations isolated after mutagenesis. We have used EcoRI to cut the genomic DNA because it provides relatively good resolution between RAD markers (Figure 1). However, any other restriction enzyme could also be used, or multiple enzymes could be used to gain increased mapping resolution. RAD mapping provides an ideal procedure to identify mutant loci when the number of gene candidates is limited.
We explored the use of RAD mapping in three different contexts. First we have performed a proof-of-principle experiment where we mapped the position of unc-13, a known mutant (Figure 3). While the numbers of RAD markers obtained was relatively low in this example (683, presumably because we experienced some sample loss), the center of the trough on chromosome I is still within 800 kb of the known position of unc-13. In the second approach, we used RAD mapping to clone the or1089ts mutant. After picking 800 F2 animals from an or1089ts/CB4856 cross, we identified those that were homozygous for the or1089ts mutation. We found a substantial enrichment of the N2 DNA on the center of chromosome I that was positioned within 276 kb from the known position of the spd-2 locus. We performed Sanger DNA sequencing on PCR products derived from the spd-2 locus and identified one sequence alteration that causes a missense mutation (Figure 4D). Thus, RAD mapping can rapidly identify candidate genes that can be further investigated by sequencing candidate genes, GIPS, or complementation tests with existing mutants. We also tested the feasibility of performing RAD mapping on F2 animals that were doubly mutant for an embryonic lethal mutation and an egg-laying defective mutant, lin-2 (present in the original mutagenized strain). Since homozygous lin-2 animals hold their embryos, it was possible to more easily and rapidly select F2 progeny homozygous for the mutation being mapped from an or1167ts; lin-2 and CB4856 cross (Figure 5). As expected, we found two regions of the genome that were enriched for N2 DNA: one corresponds to the lin-2 locus, while the second corresponds to the or1167ts mutation in the sas-6 locus. Finally, we are currently exploring the use of RAD mapping by crossing temperature-sensitive mutants to CB4856 males and allowing the progeny to reproduce at the nonpermissive temperature for many generations. This method may significantly reduce the labor in isolating the homozygosed F2 progeny and would show an exclusion of N2 DNA corresponding to the lethal locus.
In a second use of Illumina sequence technology, we have developed a genome interval pull-down method to sequence defined portions of the genome. By sequencing only intervals that contain the causal mutation, one can reduce the expenses associated with whole genome sequencing. We have successfully applied this technology to positionally clone two new mutants so far (Figure 7), as well as the control dhc-1(or195ts) mutant. We identified 45 sequence alterations in the 1.83 Mb or600sd,ts interval and 29 mutations in the 1.3 Mb or683ts interval vs. the WormBase reference sequence. Thus, if we had sequenced the entire genome of these mutants, we would have found many mutations. Therefore, it is clearly important to have some mapping data to narrow the search for the causal mutation, and RAD mapping fills this role well. As costs continue to come down, whole genome sequencing using either recombinant F2 animals (Doitsidou et al. 2010) or backcrossed mutants (Zuryn et al. 2010), two techniques that simultaneously map and sequence mutations, may become more cost effective than the relatively more labor-intensive GIPS. Nevertheless, RAD mapping may continue to prove useful for analyzing large numbers of mutants, as many mutants can be identified by sequencing only candidate genes in the vicinity or by complementation tests with previously identified alleles in the region. In fact, a large number of nonconditional mutants exist that can be used for performing complementation tests (for example, Johnsen and Baillie 1991; Clark and Baillie 1992; Stewart et al. 1998; Johnsen et al. 2000). GIPS should also remain useful for sequencing candidate genes that are too large to easily amplify with PCR for Sanger sequencing.
As of August 2011, the cost to sequence the entire C. elegans genome at 30× coverage was about U.S.$600 on the HiSeq2000 platform. With this many reads, one could perform 50 RAD mapping experiments (at 30× coverage) or ∼50 GIPS procedures using 2-Mb pull-down regions. Both of these sequencing techniques can also be run on Illumina runs with other samples (with the use of barcoded adapters). Depending on the type of mutant being sequenced, using WGS strategies will be more straightforward (and more time- and cost effective; M. Doitsidou and O. Hobert, unpublished results.) For example, if the mutant locus is resistant to RNAi, or if large-scale RNAi screens have not assayed the phenotype represented by the mutant, then WGS is likely the best approach. However, if the mutant phenotype is likely to be recapitulated by RNAi, such as early embryonic lethality (as we are studying), then RAD mapping will reveal a limited number of candidate genes. Sanger sequencing, complementation tests with existing mutants, or GIPS could then identify the causal mutation, although the time required to perform this two-step approach is longer than using a mapping/WGS approach. Thus, RAD mapping may be a viable alternative to WGS when large numbers of mutants are being investigated. Similarly, GIPS can be useful for sequencing loci that are known to be defective in many different mutants. For example, suppressor screens often identify many intragenic suppressor alleles (for example, see Greenwald and Horvitz 1980) which, depending on size, can be very time consuming to Sanger sequence; yet performing WGS may be too expensive with many alleles to sequence. GIPS fills this gap and allows the simultaneous sequencing of many different mutant loci on the same Illumina lane with the use of barcoded samples. In conclusion, we offer two new strategies for mutant identification in C. elegans that can fill roles not currently provided by WGS for certain applications.
The following additional data are available with the online version of this paper: File S1 is a table listing the genomic positions and sequence of the EcoRI-associated RAD tags present in the N2 and CB4856 strains using the WS190 referential version of WormBase.
Acknowledgments
We thank members of the Bowerman and Johnson labs for help in isolating conditional mutants and developing mapping and sequencing protocols. This work was supported by a Leukemia and Lymphoma Society of America fellowship to S.M.O. and National Institutes of Health grants GM050817 and GM049869 to B.B.
Footnotes
Supporting information is available online at http://www.genetics.org/content/suppl/2011/09/06/genetics.111.134031.DC1.
- Received June 29, 2011.
- Accepted August 25, 2011.
- Copyright © 2011 by the Genetics Society of America
