Beginning with this issue, we are introducing three new types of content that we hope will make The Worm Breeder’s Gazette more useful to you, the community of nematode researchers.  The Gazette has traditionally been a forum for the dissemination of unpublished data and methods, and we plan to continue presenting this type of content.  However, the Gazette is also a community newsletter, and we would like to enhance this function.

1). In this and subsequent issues, we will be including author-written summaries of new or improved methods from published papers.  These articles, tagged ‘Highlighted publication’, are intended to enhance the visibility of papers published as methods as well as technological advances contained in research papers.

2). Several members of the  Editorial Board of WormBook have contributed brief lists of notable recent papers in their areas of expertise.

3). You will also find announcements describing newly established worm labs and their research interests.  Subsequent issues may also include announcements of relocating labs, upcoming meetings and promotions and awards given to worm researchers.

As always, we welcome your comments and suggestions concerning The Worm Breeder’s Gazette.

We have created a library of 2,000 mutagenized C. elegans strains, each sequenced to an average depth of 15X to reveal most mutations. The library contains over 700,000 single nucleotide variants (SNVs) with, on average, 8 non-synonymous changes per gene. We generated the library using the mutagens EMS, ENU or a cocktail of EMS plus ENU. F1 populations were screened in nicotine for animals heterozygous for unc-22 mutations to ensure effectiveness of the mutagen. F2 populations were screened again to select non-unc-22 animals, and the resulting lines were selfed for a further eight generations to drive all genomic regions toward homozygosity. Whole-genome sequencing was done with paired-end reads on Illumina GAII or Hi-Seq machines using size-selected and molecularly bar-coded DNAs. Reads were aligned using Phaster (P. Green, unpublished) and SNVs were called using SamTools and custom filters. Indels and rearrangements were identified with custom tools.

Analysis of the data from the first 1,794 strains has yielded 705,748 SNPs in 20,066 genes (averaging 390 per strain). These include 159,338 non-synonymous changes in 19,449 genes (eight new alleles per gene). Of these mutations, 9,829 are knockouts (nonsense or spicing defects) in 6,774 genes, for an average of more than four per strain. Based on read numbers, the rDNA repeat copy number is surprisingly variable, with some strains having fewer than 60 copies and a few having more than 150. We have supplemented these mutagenized strains with 40 natural isolates to recover an additional 500,000 mutations. The mutation data for the first 600 mutated strains have been deposited in WormBase, with the rest of the data in process. A separate website allows direct queries of the data (http://genome.sfu.ca/mmp/). Nearly all of the 2,000 individual strains are available from the Caenorhabditis Genetics Center. We are currently building frozen kits containing all the strains in 96-well arrays, allowing parallel experimentation on a wide spectrum of mutant genes. The resource should provide rapid access to multiple mutations in any gene of interest as well as allow investigation of gene-gene interactions.

Oliver Hobert and his colleagues have pioneered the use of whole-genome sequencing (WGS) to identify lesions in C. elegans mutants, and they have produced the MAQGene software pipeline to analyze this data (Sarin et al., 2008) (http://maqweb.sourceforge.net).  While MAQGene is excellent, it runs on Linux operating systems and requires a MySQL server, and these requirements are currently beyond our (and perhaps other C. elegans researchers) computer capabilities.

We are using WGS to identify a mutation cu13 that enhances the lethal phenotype of a hypomorphic tbx-2 mutant.  Illumina sequencing was used to sequence genomic DNA of several strains that have the cu13 or wild-type alleles. Freely available software packages were used to align sequence reads with the reference C. elegans genome, identify variants, and annotate these variants with predicted effect on gene function.  These analyses identified thousands of variants in each sequenced genome, and Microsoft Access was used to sort and compare variants in each genome.  A small number of candidate lesions for cu13 were identified, and we are currently determining which of these causes the mutant phenotype.  This approach is feasible for novices like us using a desktop computer and fairly rudimentary skills with the command line interface, and we thought others in the C. elegans community might be interested in trying this for themselves.  The software packages generally have manuals and tutorials available, and we relied on these heavily.

Sequence alignment: Bowtie 2 was used to index the C. elegans reference genome and to align our fastq sequencing reads to this reference (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml).  Bowtie 2 is an ultrafast aligner that outputs a SAM (Sequence Alignment/Map) used in subsequent analyses (Langmead and Salzberg, 2012 PMID 22388286), although Bowtie 2 may be less sensitive than the MAQ aligner used in MAQGene (Nielsen et al., 2011).

Variant identification:  The SAMtools software package was used to identify variants and call genotypes based on SAM alignment files (http://samtools.sourceforge.net/) (Li et al., 2009).   SAM files were initially converted to their binary equivalent BAM format and sorted using ‘samtools view’ and ‘samtools sort’ commands.  Information regarding sequence quality and possible genotype was calculated using the ‘samtools mpileup’ command and stored in the BCF file format.  Variants were called and written to a VCF (Variant Call Format) file using the ‘bcftools view’ and ‘vcfutils.pl’ commands.  VCF is a widely used text file format storing information regarding variant position and sequence, sequence quality, and predicted genotype.

Variant annotation: C. elegans genome annotations were retrieved from the UCSC Genome Browser Annotation Database using the Perl-based software package ANNOVAR (http://www.openbioinformatics.org/annovar/) (Wang et al., 2010).  ANNOVAR was used to convert our VCF files to ANNOVAR input files and annotate variants using the ‘perl convert2annovar.pl’ and ‘perl annotate_variation.pl’ commands.  ANNOVAR outputs one file annotating all variants indicating the genomic features they hit, and a second file indicating the amino acid changes for exonic variants.  For convenience, these files were combined into a single table using Microsoft Access.

Variant and sequence visualization:  The Integrative Genomics Viewer (IGV) (http://www.broadinstitute.org/igv/home) was used to visualize variants and the underlying sequence reads (Thorvaldsdottir et al., 2012).  Variants called in VCF files and sequence alignments in BAM files can loaded into tracks in the IGV browser and can be rapidly viewed at a wide range of genomic scales.

There are a variety of software options available for each of the steps (Nielsen et al., 2011), and while the software described here works for us, we are evaluating other approaches for each of these steps.

We are interested in constructing transgenic nematodes expressing the stress-tolerance gene TPS1 from yeast (Vellai et al., 1999), which is expressed in the dauer juvenile stage both in C. elegans and Heterorhabditis bacteriophora. In C. elegans experiments we used dauer constitutive mutants DR133 and DR136, homozygous for daf-7 and daf-2 respectively, and for morphological markers unc-32 (III) and dpy-1 (III). In H. bacteriophora experiments we used a 19^th generation inbred line of H. bacteriophora strain TT01. C. elegans was grown on NGM, while H. bacteriophora was grown on ENGM (Fodor et al., The WBG 18:2). The TPS1 gene was isolated from S. cerevisiae by Z. Bánfalvi and ligated into the nematode vector (made by A. Fire) and cloned to the heat-inducible promoter hsp16-2 promoter, as described before (Vellai et al., 1999). Transformation was carried out by “bombarding.” In Hungary we have been using the Gene Booster equipment patented by Dr. B. Jenes in the Agro-Biotechnology Center, Gödöllő, Hungary. Synchronous L4 (J4) and dauer (IJ) populations were bombarded with different doses on bacterium-free NGM plates. The worms were washed off and transferred to fresh, bacterium seeded NGM (ENGM) plates. The NGM and ENGM plates were incubated in 18 and 25 ^oC, respectively. Bombarded generations considered to be P0. For C. elegans the next generation was allowed to grow and propagate on OP50-seeded NGM plates. Plates were transferred to 25 ^oC for 48h. The nematodes were washed off the plates and dauer larvae were selected in SDS. After 5X washing with sterile tap water they were heat shocked at 33 ^oC for 4h. The F1 progeny of the bombarded worms were transferred to 2.4 M NaCl for 4h. 100% of the control worms died during this time. The progeny of the bombarded worms were washed 3X and distributed to 40 NGM plates seeded with OP50 bacteria. Some survived. They are considered as potentially transformed nematodes. One individual hermaphrodite was picked up from each plate to initiate pure lines for further study. For H. bacteriophora each ENGM plate, like an island, was put in a large glass plate of sterile tap water. The lid of the ENGM plate was removed, but the large plate covered. The IJ were moving from the plates to the water. They were collected daily. They were handled similarly to C. elegans except for using 0.1 w/v hyamin instead of SDS. The Fn progeny of the bombarded worms were transferred to 2.4 M NaCl for 4h. 100% of the control died during this time. The animals were washed 3X with sterile tap water and distributed into 40 ENGM plates seeded with TT01 bacteria. Some survived. They were considered as potentially transformed nematodes. One individual hermaphrodite was picked up from each plate to initiate pure lines for further study. We found no significant difference in the rate of survival between the progeny of treated L4 (J4) and dauers.

Incorporation of unnatural amino-acids is a powerful and versatile new tool for modifying and selectively tagging proteins of interest. The technique has been shown to be applicable to C. elegans by Greiss and Chen (2011), utilizing a transgene expressing an amber-suppressing tRNA together with a cognate pyrrolysl-tRNA synthetase from Methanosarcina.

We wished to apply this technique to investigate srf-5, a gene affecting surface antigenicity and bacterial infection susceptibility, which we have found to encode a predicted small secreted protein. The reference srf-5 allele, ct115, causes an early Trp-to-opal nonsense mutation and behaves as a genetic null. We reasoned that it should be possible to rescue the mutant phenotype by introducing a transgene carrying a Trp-to-amber mutation at the same position, and crossing this transgene onto an amber-suppressing genetic background such as sup-5. Crossing the same transgene onto the unnatural-amino-acid-inserting background developed by Greiss and Chen (2011), and providing a suitable unnatural amino-acid substrate, should then result in synthesis of SRF-5* (that is, SRF-5 with an unnatural amino acid instead of Trp). If SRF-5* is as functional as wildtype SRF-5, then it should also rescue the srf-5 mutant.

Surprisingly, we found that the srf-5(amber) transgene was able to rescue the srf-5 mutant in the absence of any amber suppression, when present in a multicopy extrachromosomal array. The same effect was seen when the original opal nonsense mutant, ct115, was introduced as a multicopy extrachromosomal array. We hypothesized that transcription from the multiple copies leads to elevated srf-5 mRNA levels, and consequently to some read-through of either stop codon. We tested this interpretation by crossing srf-5(ct115) with smg-2(e2008), a mutant defective in NMD (Nonsense Mediated Decay), and found that smg-2 also partly suppressed the Srf-5 phenotype. The ct115 opal (UGA) codon therefore appears to be a leaky stop. It may be unusually leaky, or else SRF-5 may be required only in very small amounts. An alternative explanation, that the amber fragment encoded by srf-5(ct115) has some functional activity, seems unlikely because this fragment is small (<35 amino-acid residues).

Using amber transgenes for unnatural amino-acid incorporation should work for other genes, and may still work for srf-5 itself. However, the read-through effect that we report here may sometimes complicate matters.