Progress 12/01/13 to 12/31/13
Outputs
Target Audience: Aquaculture community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A total of 10 graduate students were trained. How have the results been disseminated to communities of interest? All genome information has been made available through various platforms including publications, databases, and raw data. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project is fully executed with its goals fully accomplished. We have generated a reference genome sequence assembly for catfish. The genome was annotated with supporting evidence from transcriptome analysis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Jiang Y, Gao X, Liu S, Zhang Y, Liu H, Sun F, Bao L, Waldbieser G, Liu ZJ. 2013. Whole genome comparative analysis of channel catfish (Ictalurus punctatus) with four model fish species. BMC Genomics 14:780.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Sun F, Liu S, Gao X, Jiang Y, Perera D, Wang X, Li C, Sun L, Zhang J, Kaltenboeck L, Dunham R, Liu ZJ. 2013. Male-biased genes in catfish as revealed by RNA-Seq analysis of the testis transcriptome. PLoS One 8(7):e68452.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Wang R, Feng J, Li C, Liu S, Zhang Y, Liu ZJ. 2013. Four lysozymes (one c-type and three g-type) in catfish are drastically but differentially induced after bacterial infection. Fish and Shellfish Immunology 35:136-145.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Liu S, Wang X, Sun F, Zhang J, Feng J, Liu H, Rajendran KV, Sun L, Zhang Y, Jiang Y, Kaltenboeck L., Kucuktas H, and Liu ZJ. 2013. RNA-Seq reveals expression signatures of genes involved in oxygen transport, protein synthesis, folding and degradation in response to heat stress in catfish. Physiological Genomics 45:462-476.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Zhang J, Liu S, Rajendran KV, Sun L, Zhang Y, Sun F, Kucuktas H, Liu H, Liu ZJ. 2013. Pathogen recognition receptors in channel catfish: ?. phylogeny and expression analysis of Toll-like receptors. Developmental and Comparative Immunology 40: 185-194.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Zhang Y, Liu S, Lu J, Jiang Y, Gao X, Ninwichian P, Li C, Waldbieser G, Liu ZJ. 2013. Comparative genomic analysis of catfish linkage group 8 reveals two homologous chromosomes in zebrafish with extensive inter-chromosomal rearrangements. BMC Genomics, BMC Genomics 14:387, DOI: 10.1186/1471-2164-14-387.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Liu S, Li Q, Liu ZJ. 2013. Genome-wide identification, characterization and phylogenetic analysis of 50 catfish ATP-binding cassette (ABC) transporter genes. PLoS ONE 8(5):e63895.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Jiang Y, Ninwichian P, Liu S, Zhang J, Liu H, Kucuktas H, Liu ZJ. 2013. Generation of physical contig-specific sequences useful for whole genome sequence assembly and scaffolding. PloS one 8 (10), e78872.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Sookruksawong S, Sun F, Liu ZJ, Tassanakajon A. 2013. RNA-Seq analysis reveals genes associated with resistance to Taura syndrome virus (TSV) in the Pacific white shrimp Litopenaeus vannamei. Developmental and Comparative Immunology 41:523-533.
|
Progress 12/01/09 to 12/31/13
Outputs
Target Audience: The genome research community, aquaculture research community, aquaculture industries, and general public Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project allowed training of three Ph.D. students and two postdocs How have the results been disseminated to communities of interest? All the information was placedon our webpages whenever relevant. Publications were made as rapidly as possible. RNASeq datasets were loaded to NCBI What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Overall, we have achieved and exceeded the goals set for the project. For Objective 1, we have produced the Reference channel catfish genome of high quality assembly. For Objective 2, we have produced a high quality draft genome sequence assembly rather than the original planned in silico mapping of the blue catfish sequences against the channel catfish genome assembly. Realizing that this project was conducted during a period when sequencing technologies were advancing almost every a few months and the sequencing cost was dramatically downward, we have used various sequencing technologies, the best at the time of application including Sanger sequencing for BAC end sequences, 454 sequencing, GAII Illumina sequencing (the same as now the Illumina sequencing in principle, but the reads were shorter and the efficiencies were 10s of fold less efficient), Illumina HiSeq sequencing, PacBio long reads sequencing, and Moleculo-based sequencing. Apparently, sequences generated in 2008 and 2009 were of less quality and quantity, but were much more expensive. For both channel catfish and blue catfish genomes, we have generated doubled haploid to reduce assembly complexities, which has been proven to be the right decision. This project continually incorporated new sequencing technologies and bioinformatic algorithms. Interactions with sequencing providers and software developers have been mutually beneficial. Doubled haploid channel catfish were produced by pressure- or thermal-induced disruption of the first mitotic cleavage in fertilized catfish eggs. Microsatellite-based genotyping confirmed genomic homozygosity on all chromosomes and one individual was chosen as the genome donor. Initially, the project explored 454 sequencing technology as the basis for genome sequence production. We produced 1.6M sequencing reads (518Mb total, average read length 321 bp). Initial analysis demonstrated that a high proportion of sequences were significantly shortened because the template contained a short tandem repeat. Therefore, the project was shifted to the Illumina platform. Paired end Illumina sequences were produced from randomly sheared fragments (approximately 400 bp insert size) and Nextera-sheared fragments (approximately 250 bp insert size). Paired end sequences were also produced from three Illumina mate-paired libraries with average insert sizes of 3kb, 7kb, and 36kb. For sequence processing and assembly, we first used the ABySS assembly software on the Amazon Elastic Compute Cloud. Interaction with the ABySS developers led to improvements in their software and a protocol for implementing ABySS on the Amazon cloud. We subsequently gained access to the High Performance Computing Center at Mississippi State University through their Institute for Genomics Biocomputing and Biotechnology. The best ABySS assembly consisted of 256,408 contigs with an N50 contig length of 5,588 bp. Despite a significant amount of trial and interaction with the developers, ABySS would not incorporate the mate paired DNA sequences to produce scaffolds in this large genome. Near the end of this project, third generation DNA sequencing technology had matured to the stage where we utilized grant funding to purchase an 11-fold genome coverage of Pacific Biosciences (PacBio) long read DNA sequence via a commercial vendor. More than 3 million sequences were produced with an average length > 3kb, but the sequence quality of that generation of the technology still contained random sequencing error that would inhibit efficient assembly. An error correction algorithm based on the Celera Assembler (pacBioToCA) became available but the size of our PacBio and Illumina data sets exceeded our bioinformatic capacity. Therefore we cooperated with the algorithm’s developer at the Dept. of Homeland Security to correct the PacBio sequences using our high-quality Illumina sequences. This work has only recently been completed and has produced 3.3 million error-corrected sequences totaling 5,424 Mb with an average length of 1,636 bp. Our next approach was to utilize the Whole Genome Assembler (Celera Assembler) which could incorporate multiple types of sequence. This required the purchase of a Linux-based bioinformatics server with 64 processors, 512GB RAM, and 12TB of disk storage (which has been upgraded to 22 TB). Our research coincided with the development of MaSuRCA, an assembly pipeline based on Celera Assembler. Our interaction with the developers has contributed to improvements in the MaSuRCA software and has led to the first generation assembly of the channel catfish genome. The assembly was based primarily on Illumina paired sequencing reads from the short insert and long insert libraries. Gaps in the assembled scaffolds were closed using a combination of Illumina and corrected PacBio sequences in the MaSuRCA pipeline. Changing the assembly algorithm from ABySS to MaSuRCA significantly improved the assembly. The current version of the catfish genome assembly (v2.2.0) is contained in only 40% of the number of ABySS contigs and 90% of the assembly is contained in only 24% of the number of ABySS contigs. 90% of the channel catfish genome sequences are included in 29,238 contigs; and 95% of the channel catfish genome sequences are included in 46,936 contigs. MaSuRCA produced 822 Mb of scaffolded bases, and 90% of the assembled bases are contained in only 4,280 scaffolds. Half of the assembly is contained in only 51 scaffolds ranging from 4.3 Mb to 15.5 Mb in length. Addition of PacBio sequence to the MaSuRCA gap closing pipeline led to closure of 19,284 gaps in the scaffolds. To convert such high quality assembly of the draft genome into chromosome-based Reference genome, a high-density SNP chip with 250,000 SNPs was constructed to map the SNPs to the catfish genome. With over 65,000 SNPs selected for map construction, 29 linkage groups were constructed using a LOD threshold value of 8.0. The sex-averaged map consisted of 29,081 markers and spanned 3,505 cM with an averaged map interval of 0.38 cM/marker. The female map consisted of 18,444 SNPs in total, with a total map length of 4,495 cM. The averaged female map interval was 0.64 cM/marker. The male map comprised 15,148 markers and covered 2,593 cM with an average marker interval of 0.62 cM. In spite of such terrific progress, the catfish 250K SNP arrays were constructed before the genome was assembled. Therefore, there are still a large number co contigs that are not represented by the array, and therefore, they are not mapped to linkage groups yet. We are in the early phase of planning for the construction of a 675K SNP array based on the whole genome assembly. The catfish genome was annotated using transcriptome sequencing. Through transcriptome analysis of various tissues, a total of 27,953 genes have been identified, of which over 23,000 complete cDNAs have been assembled and annotated. Gene families and gene duplication were analyzed. Doubled haploid blue catfish were produced using the same methods as for channel catfish, and we used microsatellite genotyping to verify genome homozygosity in the donor fish. We initiated a collaboration with Moleculo, Inc. (now Illumina) to produce long, high quality sequencing reads via their proprietary technology. We have produced 6,470 Mb in 1.4M Moleculo sequences, with an average length of 4,653 bp. The Moleculo process also has provided scaffolding information for 33,644 pairs of sequences. Our presentation of the data at the Plant and Animal Genome Conference 21 (January 2013 and also January 2014) was the first public presentation of Moleculo technology. Such high quality sequences allowed assembly of the draft blue catfish genome.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Jiang Y, Ninwichian P, Liu S, Zhang J, Liu H, Kucuktas H, Liu ZJ. 2013. Generation of physical contig-specific sequences useful for whole genome sequence assembly and scaffolding. PloS one 8 (10), e78872.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Liu S, Li Q, Liu ZJ. 2013. Genome-wide identification, characterization and phylogenetic analysis of 50 catfish ATP-binding cassette (ABC) transporter genes. PLoS ONE 8(5):e63895. doi:10.1371/journal.pone.0063895.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Zhang Y, Liu S, Lu J, Jiang Y, Gao X, Ninwichian P, Li C, Waldbieser G, Liu ZJ. 2013. Comparative genomic analysis of catfish linkage group 8 reveals two homologous chromosomes in zebrafish with extensive inter-chromosomal rearrangements. BMC Genomics, BMC Genomics 14:387, DOI: 10.1186/1471-2164-14-387.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Sun F, Ninwichian P, Liu SK, Liu, ZJ. 2013. Identification and application of sex-specific markers in catfish. In: Sex determination in fishes, ed. Songlin Chen, pp. 131-139, Academic Press, Beijing.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Zhang J, Liu S, Rajendran KV, Sun L, Zhang Y, Sun F, Kucuktas H, Liu H, Liu ZJ. 2013. Pathogen recognition receptors in channel catfish: ?. phylogeny and expression analysis of Toll-like receptors. Developmental and Comparative Immunology 40: 185-194.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Liu S, Wang X, Sun F, Zhang J, Feng J, Liu H, Rajendran KV, Sun L, Zhang Y, Jiang Y, Kaltenboeck L., Kucuktas H, and Liu ZJ. 2013. RNA-Seq reveals expression signatures of genes involved in oxygen transport, protein synthesis, folding and degradation in response to heat stress in catfish. Physiological Genomics 45:462-476.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Wang R, Feng J, Li C, Liu S, Zhang Y, Liu ZJ. 2013. Four lysozymes (one c-type and three g-type) in catfish are drastically but differentially induced after bacterial infection. Fish and Shellfish Immunology 35:136-145.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Preziosa E, Liu S, Gao X, Liu H, Kucuktas H, Terova G, and Liu ZJ. 2013. Effect of Nutrient Restriction and Re-feeding on Calpain Family Genes in Skeletal Muscle of Channel Catfish (Ictalurus punctatus). PLoS One 8:e59404.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Sun F, Liu S, Gao X, Jiang Y, Perera D, Wang X, Li C, Sun L, Zhang J, Kaltenboeck L, Dunham R, Liu ZJ. 2013. Male-biased genes in catfish as revealed by RNA-Seq analysis of the testis transcriptome. PLoS One 8(7):e68452. doi:10.1371/journal.pone.0068452.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Jiang Y, Gao X, Liu S, Zhang Y, Liu H, Sun F, Bao L, Waldbieser G, Liu ZJ. 2013. Whole genome comparative analysis of channel catfish (Ictalurus punctatus) with four model fish species. BMC Genomics 14:780. DOI: 10.1186/1471-2164-14-780. This paper was highlighted in The Scientist http://www.the-scientist.com//?articles.view/articleNo/38334/title/Genome-Digest/
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Wang R, Sun L, Bao L, Zhang J, Jiang Y, Yao J, Song L, Feng J, Liu S, Liu ZJ. 2013. Bulk segregant RNA-seq reveals expression and positional candidate genes and allele-specific expression for disease resistance against enteric septicemia of catfish. BMC Genomics 14: 929. DOI: 10.1186/1471-2164-14-929
|
Progress 12/01/12 to 11/30/13
Outputs
Target Audience: The aquaculture research community, aquaculture industry and all animal scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project allowed training of six graduate students. How have the results been disseminated to communities of interest? All the information has been made available immediately after accuratedocumentation through publications, databases, and raw data presentations. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Overall, we have achieved and exceeded the goals set for the project. For Objective 1, we have produced the Reference channel catfish genome rather than the original planned draft genome assembly. For Objective 2, we have produced a high quality draft genome sequence assembly rather than the original planned in silico mapping of the blue catfish sequences against the channel catfish genome assembly. Realizing that this project was conducted during a period when sequencing technologies were advancing almost every a few months and the sequencing cost was dramatically downward, we have used various sequencing technologies, the best at the time of application including Sanger sequencing for BAC end sequences, 454 sequencing, GAII Illumina sequencing (the same as now the Illumina sequencing in principle, but the reads were shorter and the efficiencies were 10s of fold less efficient), Illumina HiSeq sequencing, PacBio long reads sequencing, and Moleculo-based sequencing. Apparently, sequences generated in 2008 and 2009 were of less quality and quantity, but were much more expensive. For both channel catfish and blue catfish genomes, we have generated doubled haploid to reduce assembly complexities, which has been proven to be the right decision. This project continually incorporated new sequencing technologies and bioinformatic algorithms. Interactions with sequencing providers and software developers have been mutually beneficial. Initially, the project explored 454 sequencing technology as the basis for genome sequence production. We produced 1.6M sequencing reads (518Mb total, average read length 321 bp). Initial analysis demonstrated that a high proportion of sequences were significantly shortened because the template contained a short tandem repeat. Therefore, the project was shifted to the Illumina platform. Paired end Illumina sequences were produced from randomly sheared fragments (approximately 400 bp insert size) and Nextera-sheared fragments (approximately 250 bp insert size). Paired end sequences were also produced from three Illumina mate-paired libraries with average insert sizes of 3kb, 7kb, and 36kb. For sequence processing and assembly, we first used the ABySS assembly software on the Amazon Elastic Compute Cloud. Interaction with the ABySS developers led to improvements in their software and a protocol for implementing ABySS on the Amazon cloud. We subsequently gained access to the High Performance Computing Center at Mississippi State University through their Institute for Genomics Biocomputing and Biotechnology. The best ABySS assembly consisted of 256,408 contigs with an N50 contig length of 5,588 bp. Despite a significant amount of trial and interaction with the developers, ABySS would not incorporate the mate paired DNA sequences to produce scaffolds in this large genome. Near the end of this project, third generation DNA sequencing technology had matured to the stage where we utilized grant funding to purchase an 11-fold genome coverage of Pacific Biosciences (PacBio) long read DNA sequence via a commercial vendor. More than 3 million sequences were produced with an average length > 3kb, but the sequence quality of that generation of the technology still contained random sequencing error that would inhibit efficient assembly. An error correction algorithm based on the Celera Assembler (pacBioToCA) became available but the size of our PacBio and Illumina data sets exceeded our bioinformatic capacity. Therefore we cooperated with the algorithm’s developer at the Dept. of Homeland Security to correct the PacBio sequences using our high-quality Illumina sequences. This work has only recently been completed and has produced 3.3 million error-corrected sequences totaling 5,424 Mb with an average length of 1,636 bp. Our next approach was to utilize the Whole Genome Assembler (Celera Assembler) which could incorporate multiple types of sequence. This required the purchase of a Linux-based bioinformatics server with 64 processors, 512GB RAM, and 12TB of disk storage (which has been upgraded to 22 TB). Our research coincided with the development of MaSuRCA, an assembly pipeline based on Celera Assembler. Our interaction with the developers has contributed to improvements in the MaSuRCA software and has led to the first generation assembly of the channel catfish genome. The assembly was based primarily on Illumina paired sequencing reads from the short insert and long insert libraries. Gaps in the assembled scaffolds were closed using a combination of Illumina and corrected PacBio sequences in the MaSuRCA pipeline. Changing the assembly algorithm from ABySS to MaSuRCA significantly improved the assembly (Table 1). The current version of the catfish genome assembly (v1.1) is contained in only 40% of the number of ABySS contigs and 90% of the assembly is contained in only 24% of the number of ABySS contigs. 95% of the channel catfish genome sequences are included in 46,936 contigs. MaSuRCA produced 822 Mb of scaffolded bases, and 90% of the assembled bases are contained in only 3,903 scaffolds. Half of the assembly is contained in only 58 scaffolds ranging from 3.8Mb to 15.5Mb in length. Addition of PacBio sequence to the MaSuRCA gap closing pipeline led to closure of 19,284 gaps in the scaffolds. To convert such high quality assembly of the draft genome into chromosome-based Reference genome, a high-density SNP chip with 250,000 SNPs was constructed to map the SNPs to the catfish genome. With over 65,000 SNPs selected for map construction, 29 linkage groups were constructed using a LOD threshold value of 8.0. The sex-averaged map consisted of 35,178 markers and spanned 7,088.5 cM with an averaged map interval of 0.38 cM/marker. The female map consisted of 21,967 SNPs in total, with a total map length of 7,502.7 cM. The averaged female map interval was 0.64 cM/marker. The male map comprised 17,934 markers and covered 5,421.7 cM with an average marker interval of 0.62 cM. A total of 86% of the catfish scaffolds from catfish genome assembly were anchored onto the 29 chromosomes. The catfish genome was annotated using transcriptome sequencing. Through transcriptome analysis of various tissues, a total of over 23,000 complete cDNAs have been assembled and annotated. Gene families and gene duplication were analyzed. Blue catfish genome sequencing. Doubled haploid blue catfish were produced using the same methods as for channel catfish, and we used microsatellite genotyping to verify genome homozygosity in the donor fish. We initiated a collaboration with Moleculo, Inc. (now Illumina) to produce long, high quality sequencing reads via their proprietary technology. We have produced 6,470 Mb in 1.4M Moleculo sequences, with an average length of 4,653 bp. The Moleculo process also has provided scaffolding information for 33,644 pairs of sequences. Our presentation of the data at the Plant and Animal Genome Conference 21 (January 2013) was the first public presentation of Moleculo technology. Such high quality sequences allowed assembly of the draft blue catfish genome.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
4. Wang R, Sun L, Bao L, Zhang J, Jiang Y, Yao J, Song L, Feng J, Liu S, Liu ZJ. 2013. Bulk segregant RNA-seq reveals expression and positional candidate genes and allele-specific expression for disease resistance against enteric septicemia of catfish. BMC Genomics BMC Genomics 14: 929.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
5. Feng J, Liu S, Wang X, Kaltenboeck L, Kucuktas H, Li J, Liu ZJ 2014. Hemoglobin genes are differentially regulated under heat stress conditions between sensitive and tolerant catfish in different tissues. Comparative Biochemistry and Physiology, Part D, Genomics and Proteomics 9:11-22.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
6. Dunham R, Taylor JF, Rise M, Liu ZJ, 2013. Development of strategies for integrated breeding, genetics and applied genomics for genetic improvement of aquatic organisms. Aquaculture, in press.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
1. Geng X, Feng J, Liu S, Wang Y, 2, Arias C, Liu ZJ. 2014. Transcriptional regulation of hypoxia inducible factors alpha (HIF-alpha) and their inhibiting factor (FIH-1) of channel catfish (Ictalurus punctatus) under hypoxia. Comparative Biochemistry and Physiology, Part B, Biochemistry and Molecular Biology, in press.
|
Progress 12/01/11 to 11/30/12
Outputs
OUTPUTS: This project has two objectives: 1). To sequence, assemble, and annotate the reference channel catfish genome de novo, using a homozygous DNA template and a combination of new 2nd generation sequences and existing Sanger sequences to achieve a high quality, cost-efficient assembly; and 2) Efficiently produce and annotate the reference blue catfish genome by in silico mapping of 2nd generation sequences from a homozygous blue catfish to the reference channel catfish genome sequence assembly. Channel catfish genome sequencing: As we reported last year, sequences of channel catfish genome are now under analysis for the final assembly. Blue catfish genome sequencing: Doubled haploid blue catfish were produced using the same methods as for channel catfish. Only two homozygous blue catfish survived, and the fish selected as the genome sequence donor displayed homozygosity for maternal alleles. With the experience and lessons learned from sequencing the channel catfish genome, we has taken a novel strategy for the blue catfish genome using the Moleculo's Long Reads product to generate extremely long and accurate reads. Moleculo's library prep shears and modifies genomic DNA by adding custom DNA tags, clonally amplifying those fragments and converting them into standard short read sequencing libraries. These libraries are sequenced on an Illumina HiSeq 2000. After sequencing, a commercial algorithm is used for reconstruction and base-calling of the original fragments. This approach minimizes the presence of more than one interspersed repeat in each sub-library, thus making the genomic repeat "unique" in the sub-library. Therefore the assembly algorithm is now capable of assembling an interspersed repeat in the context of unique flanking sequence. To date, two libraries have been run on one HiSeq 2000 lane each to produce 201,508 long reads (933Mb). Eighty percent of the total sequence was found in 119,407 long reads of at least 3,340 bp. In order to test contiguity of long reads, pairwise alignments revealed 145,189 long reads contained only one or no mismatched bases along a length of 400-15,887bp. A preliminary assembly with only the long reads using 99% sequence overlap identity produced 46,098 contigs with an N50 length of 12.9 kb and N80 length of 8.5 kb. A further 42,141 long reads remained singlets with an N80 length of 4.6kb and N50 length of 7.0kb. In order to determine whether known repetitive sequences were assembled with flanking sequence, long reads were aligned with the 1.6 kb Tip1 and 1009 bp Tip2 transposons of channel catfish. Twenty one long reads contained an average 2.1 kb (minimum 200 bp) of sequence flanking the Tip1 ortholog. Similarly, 226 long reads contained an average 3.0 kb (minimum 200 bp) of sequence flanking the Tip2 orthologs. The initial results demonstrate the utility of long and accurate DNA reads in bridging repetitive regions of the genome that cannot be otherwise resolved. Four additional Moleculo Long Read libraries are currently being sequenced, to produce an additional ~2 Gb of sequence in long reads. These sequences will serve as the backbone of a genome sequence assembly for the blue catfish. PARTICIPANTS: The entire catfish research community TARGET AUDIENCES: The entire catfish research community PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This project is a historical milestone for catfish research. Once regarded as the utopia, sequencing the catfish entire genome was made possible by recent advances of sequencing technologies. We finally have a thorough structural understanding of the entire catfish genome. Along with many of the SNPs we have identified, genes identified, this project will allow us to annotate the vast majority, if not all, catfish genes to chromosomes. It allowed us to generate the large numbers of SNP markers that can be used for any genetic analysis. The catfish research community has always been limited with molecular markers that are in large numbers and cover the entire genome, and this bottleneck is finally resolved. We believe that this project has now set the stage to study any performance and production trait. The SNP-based array is being developed, and that will bring the catfish genome research one more step toward having a real world impact. We have already generated markers linked with sex, and many markers that are linked with performance traits are now being discovered. The large set of transcripts reconstructed in this study will provide the much needed resources for functional genome research in catfish, serving as a reference transcriptome for studying gene duplication and gene family structures, digital gene expression analysis, as well as aiding in the annotation of the catfish genome. Furthermore, the full set of transcripts with "signs" of SNPs has been identified, which may represent transcripts from duplicated gene copies in the genome. Therefore, this work will also lay ground for genome-scale analysis of gene duplication.
Publications
- Sun F, Peatman E, Li C, Liu S, Jiang Y, Zhou Z, Liu ZJ. 2012. Transcriptomic signatures of attachment, NF-κB suppression and IFN stimulation in the catfish gill following columnaris bacterial infection. Developmental and Comparative Immunology 38: 169-180.
- Wang R, Li C, Stoeckel J, Moyer G, Liu ZJ, and Peatman E. 2012. Rapid development of molecular resources for a freshwater mussel, Villosa lienosa (Bivalvia: Unionidae) using a RNA-seq-based approach. Freshwater Science (formerly Journal of the North American Benthological Society) 31: 695-708.
- Ninwichian P, Peatman E, Liu H, Kucuktas H, Somridhivej B, Liu S, Li P, Jiang Y, Sha Z, Kaltenboeck M, Abernathy J, Wang W, Chen F, Lee Y, Wong L, Wang S, Lu J, Liu ZJ. 2012. Second generation genetic linkage map and its integration with the BAC-based physical map in channel catfish. G3: Genes, Genomes, Genetics 10: 1233-1241.
- Lu J, Peatman E, Tang H, Lewis J, Liu ZJ. 2012. Profiling of gene duplication patterns of teleost genomes: Evidence for rapid lineage-specific genome expansion mediated by recent tandem duplications. BMC Genomics 13:246.
- Ninwichian P, Peatman E, Perera D, Liu S, Kucuktas H, Dunham R, and Liu Z.J. 2012. Identification of a sex-linked marker for channel catfish. Animal Genetics 43:476-477.
- Li C, Zhang Y, Wang R, Lu J, Nandi S, Mohanty S, Terhune J, Liu ZJ, Peatman E. 2012. RNA-Seq analysis of mucosal immune responses reveals signatures of intestinal barrier disruption and pathogen entry following Edwardsiella ictaluri infection in channel catfish, Ictalurus punctatus. Fish & Shellfish Immunology 32:816-827.
- Rajendran KV, Zhang J, Liu S, Kucuktas H, Wang X, Liu H, Wood T, Terhune J, Peatman E, Liu ZJ. 2012. Pathogen recognition receptors in channel catfish: II. Identification, phylogeny and expression of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), Developmental and Comparative Immunology 37:381-389.
- Browdy, C.L., Hulata, G., Liu, Z., Allan, G.L., Sommerville, C., Passos de Andrade, T., Pereira, R., Yarish, C., Shpigel, M., Chopin, T., Robinson, S., Avnimelech, Y. & Lovatelli, A. 2012. Novel and emerging technologies: can they contribute to improving aquaculture sustainability In R.P. Subasinghe, J.R. Arthur, D.M. Bartley, S.S. De Silva, M. Halwart, N. Hishamunda, C.V. Mohan & P. Sorgeloos, eds. Farming the Waters for People and Food. Proceedings of the Global Conference on Aquaculture 2010, Phuket, Thailand. 22-25 September 2010. pp. 149-191. FAO, Rome and NACA, Bangkok.
- Zhang H, Peatman E, Liu H, Feng T, Chen L, Liu ZJ. 2012. Molecular characterization of three L-type lectin genes from channel catfish, Ictalurus punctatus and their responses to Edwardsiella ictaluri challenge. Fish and Shellfish Immunology 32:598-608.
- Rajendran KV, Zhang J, Liu Shikai, Kucuktas H, Wang X, Liu H, Sha Z, Terhune J, Peatman E, Liu ZJ. 2012. Pathogen recognition receptors in channel catfish: I. Identification, phylogeny and expression of NOD-like receptors. Developmental and Comparative Immunology 37:77-86.
- Liu, S, Zhang Y, Sun F, Jiang Y, Wang R, Li C, Zhang J, Liu ZJ. 2012. Approaches for Functional Genomics in aquaculture. pp. 1-40. In: Functional Genomics in Aquaculture, Wiley and Blackwell Publishing, Ames, IA.
- Wang S and Liu ZJ. 2010. SNP discovery through EST data mining. pp. 91-108. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
- Wang S, Liu H, and Liu ZJ. 2010. SNP quality assessment. pp. 109-122. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
- Lu J, Peatman E, Yang Q, Wang S, Hu Z, Kucuktas H, Liu ZJ. 2010. Catfish Genome Database cBARBEL: A informatic platform for genome biology of ictalurid catfish. Nucleic Acids Research 2010: 1-7,
- Liu ZJ, Peatman E. 2010. Whole Genome Sequencing of Aquaculture Species: Identifying the Genotypes behind Production Phenotypes. Global Aquaculture Advocate, March/April 2010:68-70.
- Liu H, Takano T, Peatman E, Abernathy J, Wang S, Sha Z, Kucuktas H, Liu ZJ. 2010. Molecular characterization and gene expression of the channel catfish ferritin H subunit after bacterial infection and iron treatment. Journal of Experimental Zoology, Part A: Ecological Genetics and Physiology 313:359-368.
- Jiang Y, Abernathy J, Peatman E, Liu H, Wang S, Xu D-H, Kucuktas H, Klesius P, Liu Z.J. 2010. Identification and characterization of matrix metalloproteinase-13 sequence structure and expression during embryogenesis and infection in channel catfish (Ictalurus punctatus). Developmental and Comparative Immunology 34:590-597.
- Liu H, Jiang Y, Wang S, Ninwichian P, Somridhivej B, Xu P, Abernathy J, Kucuktas H, Liu ZJ. 2009. Comparative analysis of catfish BAC end sequences with the zebrafish genome. BMC Genomics10:592.
- Chen F, Lee Y, Jiang Y, Wang S, Peatman E, Abernathy J, Liu H, Liu S, Kucuktas H, Ke C, Liu ZJ. 2010. Identification and characterization of full-length cDNAs in catfish (Ictalurus spp.). PLoS One 12, e11546.
- Lu J, Peatman E, Wang W, Yang Q, Abernathy J, Wang S, Kucuktas H, Liu ZJ. 2010. Alternative splicing in teleost fish genomes: Same-species and cross-species analysis and comparisons. Molecular Genetics and Genomics 283:531-539.
- Zhang J, Jiang Y, Wang R, Li C, Zhang Y, Sun F, Liu, S, Liu ZJ. 2012. Chapter 2, Genomic resources for aquaculture functional genomics. pp. 41-77. In: Functional Genomics in Aquaculture, Wiley and Blackwell Publishing, Ames, IA.
- Zhou Z, Liu H, Liu S, Sun F, Peatman E, Kucuktas H, Kaltenboeck L, Feng T, Zhang H, Niu D, Lu J, Waldbieser G, Liu ZJ. 2012. Alternative complement pathway of channel catfish (Ictalurus punctatus): molecular characterization, mapping and expression analysis of factors Bf/C2 and Df. Fish and Shellfish Immunology 32:186-195.
- Zhang H, Peatman E, Liu H, Niu D, Feng T, Kucuktas H, Waldbieser G, Chen L, Liu ZJ. 2012. Characterization of mannose-binding lectin from channel catfish (Ictalurus punctatus). Research in Veterinary Science 92:408-413.
- Jiang Y, Lu J, Peatman E, Kucuktas H, Liu S, Wang S, Sun F, Liu ZJ. 2011. A Pilot study for channel catfish whole genome sequencing and de novo assembly. BMC Genomics 12:629 doi:10.1186/1471-2164-12-629.
- Feng T, Zhang H, Liu H, Zhou Z, Niu D, Wong L, Kucuktas H, Liu X, Peatman E, Liu ZJ. 2011. Molecular characterization and expression analysis of the channel catfish cathepsin D genes. Fish and Shellfish Immunology 31:164-169.
- Niu D, 1, Peatman E, Liu H, Lu J, Kucuktas H, Liu S, Sun F, Zhang H, Feng T, Zhou Z, Terhune J, Waldbieser G, Li J, Liu ZJ. 2011. Microfibrillar-associated protein 4 (MFAP4) genes in catfish play a novel role in innate immune responses. Developmental and Comparative Immunology 35:568-579.
- Liu ZJ. 2011. Development of genomic resources in support of sequencing, assembly, and annotation of the catfish genome. Comparative Biochemistry and Physiology, Part D, Genomics and Proteomics 6:11-17. doi:10.1016/j.cbd.2010.03.001.
- Liu ZJ. 2010. Genomic variations, marker technologies and their usefulness for genome-based selection. pp. 3-20. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
- Lu J and Liu ZJ. 2010. Copy number variations (CNV) and whole genome selection. pp. 21-34. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
- Kucuktas H and Liu ZJ. 2010. Construction of libraries for next generation sequencing. pp. 57-68. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
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Progress 12/01/10 to 11/30/11
Outputs
OUTPUTS: First, genomic DNA from the homozygous donor was sheared using a Hydroshear instrument and processed for 454 single read sequencing. Sequences were assembled using Newbler. Initial analysis of ~ 1 Gb of data showed that read length and contig length were significantly reduced due to the inability of the sequencing chemistry to efficiently traverse short tandem repeats. Based on the analysis, we decided that Illumina paired end sequencing would be more cost efficient and would permit a greater level of sequence contiguity and scaffolding. Second, Covaris random shearing or Nextera transposon insertion (Epicentre Technologies) libraries were produced from 400-500 bp fragments and were sequenced on an Illumina GAIIx instrument. The Covaris library produced 118.4 million pairs of 120 bp reads and the Nextera library produced 207.0 million pairs of 150 bp reads, which resulted in approximately 60-fold genome coverage. Assembly of these paired end sequences using the ABySS (1.2.7) algorithm produced 213,728 contigs covering 754.8 Mb. This level of genomic coverage was within our expectations because this assembly would not have included the repetitive regions of the genome. Analysis of the assembled Illumina-based contigs demonstrated a high level of fidelity with existing Sanger sequence from channel catfish. Therefore we proceeded with Illumina mate-pair sequencing in order to begin scaffolding the contigs. Third, mate pair libraries were produced from the reference catfish to include fragments of approximately 3kb and 8 kb using the Illumina mate pair library kit. A fosmid library containing 34-38 kb inserts was also produced from the reference catfish, and mate-paired sequences were produced by batch shearing of fosmid clones to 8-10 kb, religation of sheared DNA, then amplification of the re-joined inserts using vector based primers. A first round of 100 bp paired read sequencing has produced 5.2M paired reads from 3kb fragments, 29.2 M paired reads from 8 kb fragments, and 20.9 M paired reads from 34-36 kb fragments. These sequences are currently being added to the contigs using ABySS to assess the extent of coverage and level of scaffolding, and the potential need for additional libraries. Fourth, we also initiated an alternate approach to supplement the Illumina and Sanger sequence data. DNA libraries were produced from the reference catfish by random shearing to 8-14 kb and were shipped to Expression Analysis, Inc. (Raleigh, NC) for single molecule real-time sequencing using the Pacific Biosciences RA platform. We have contracted with this vendor to produce 6.6 Gb of sequence, approximately 6-fold genome coverage. As of 11/28/2011, ~1.6 Gb of sequence has been produced with an average read length of 3,200 bp. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This project is a historical milestone for catfish research. Once regarded as the utopia, sequencing the catfish entire genome was made possible by recent advances of sequencing technologies. We finally have a thorough structural understanding of the entire catfish genome. Along with many of the SNPs we have identified, genes identified, this project will allow us to annotate the vast majority, if not all, catfish genes to chromosomes. It allowed us to generate the large numbers of SNP markers that can be used for any genetic analysis. The catfish research community has always been limited with molecular markers that are in large numbers and cover the entire genome, and this bottleneck is finally resolved. We believe that this project has now set the stage to study any performance and production trait. The SNP-based array is being developed, and that will bring the catfish genome research one more step toward having a real world impact. We have already generated markers linked with sex, and many markers that are linked with performance traits are now being discovered. The large set of transcripts reconstructed in this study will provide the much needed resources for functional genome research in catfish, serving as a reference transcriptome for studying gene duplication and gene family structures, digital gene expression analysis, as well as aiding in the annotation of the catfish genome. Furthermore, the full set of transcripts with "signs" of SNPs has been identified, which may represent transcripts from duplicated gene copies in the genome. Therefore, this work will also lay ground for genome-scale analysis of gene duplication.
Publications
- Feng T, Zhang H, Liu H, Zhou Z, Niu D, Wong L, Kucuktas H, Liu X, Peatman E, Liu ZJ. 2011. Molecular characterization and expression analysis of the channel catfish cathepsin D genes. Fish and Shellfish Immunology 31:164-169.
- Zhang H, Peatman E, Liu H, Niu D, Feng T, Kucuktas H, Waldbieser G, Chen L, Liu ZJ. 2011. Characterization of mannose-binding lectin from channel catfish (Ictalurus punctatus). Research in Veterinary Science, in press.
- Niu D, 1, Peatman E, Liu H, Lu J, Kucuktas H, Liu S, Sun F, Zhang H, Feng T, Zhou Z, Terhune J, Waldbieser G, Li J, Liu ZJ. 2011. Microfibrillar-associated protein 4 (MFAP4) genes in catfish play a novel role in innate immune responses. Developmental and Comparative Immunology 35:568-579.
- Liu ZJ. 2011. Development of genomic resources in support of sequencing, assembly, and annotation of the catfish genome. Comparative Biochemistry and Physiology, Part D, Genomics and Proteomics 6:11-17. doi:10.1016/j.cbd.2010.03.001.
- Liu ZJ. 2010. Genomic variations, marker technologies and their usefulness for genome-based selection. pp. 3-20. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
- Lu J and Liu ZJ. 2010. Copy number variations (CNV) and whole genome selection. pp. 21-34. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
- Kucuktas H and Liu ZJ. 2010. Construction of libraries for next generation sequencing. pp. 57-68. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
- Wang S and Liu ZJ. 2010. SNP discovery through EST data mining. pp. 91-108. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
- Wang S, Liu H, and Liu ZJ. 2010. SNP quality assessment. pp. 109-122. In: Next Generation Sequencing, SNPs, and Whole Genome Selection, Wiley and Blackwell Publishing, Ames, IA.
- Lu J, Peatman E, Yang Q, Wang S, Hu Z, Kucuktas H, Liu ZJ. 2010. Catfish Genome Database cBARBEL: A informatic platform for genome biology of ictalurid catfish. Nucleic Acids Research 2010: 1-7, doi:10.1093/nar/gkq765. Chen F, Lee Y, Jiang Y, Wang S, Peatman E, Abernathy J, Liu H, Liu S, Kucuktas H, Ke C, Liu ZJ. 2010. Identification and characterization of full-length cDNAs in catfish (Ictalurus spp.). PLoS One 12, e11546.
- Lu J, Peatman E, Wang W, Yang Q, Abernathy J, Wang S, Kucuktas H, Liu ZJ. 2010. Alternative splicing in teleost fish genomes: Same-species and cross-species analysis and comparisons. Molecular Genetics and Genomics 283:531-539.
- Liu ZJ, Peatman E. 2010. Whole Genome Sequencing of Aquaculture Species: Identifying the Genotypes behind Production Phenotypes. Global Aquaculture Advocate, March/April 2010:68-70.
- Liu H, Takano T, Peatman E, Abernathy J, Wang S, Sha Z, Kucuktas H, Liu ZJ. 2010. Molecular characterization and gene expression of the channel catfish ferritin H subunit after bacterial infection and iron treatment. Journal of Experimental Zoology, Part A: Ecological Genetics and Physiology 313:359-368.
- Jiang Y, Abernathy J, Peatman E, Liu H, Wang S, Xu D-H, Kucuktas H, Klesius P, Liu Z.J. 2010. Identification and characterization of matrix metalloproteinase-13 sequence structure and expression during embryogenesis and infection in channel catfish (Ictalurus punctatus). Developmental and Comparative Immunology 34:590-597.
- Liu H, Jiang Y, Wang S, Ninwichian P, Somridhivej B, Xu P, Abernathy J, Kucuktas H, Liu ZJ. 2009. Comparative analysis of catfish BAC end sequences with the zebrafish genome. BMC Genomics10:592.
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Progress 12/01/09 to 11/30/10
Outputs
OUTPUTS: The catfish whole genome sequencing project is well under way. To date 50X genome coverage of Illumina sequences have been generated. Sequencing using 454 and other platforms is under way. It is anticipated that the whole genome will be sequenced and draft genome sequences be assembled in a few months. Simultaneously, several lines of research activities are being conducted to support the assembly and annotation of the whole genome sequence including 1) mapping of BAC end-associated microsatellites to linkage maps to integrate the genetic and physical maps; and 2) transcriptome sequencing to generate gene-associated SNPs; and 3) large scale SNP identification through sequencing of reduced representation libraries. To date, about 2,000 BAC end-associated microsatellites were mapped to the linkage map. The genotyping has been completed, and the work is being prepared for publication. PARTICIPANTS: all catfish genome community TARGET AUDIENCES: The catfish genome community and catfish researchers, industry groups. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Catfish Genome Database cBARBEL (abbreviated from catfish Breeder and Researcher Bioinformatics Entry Location) has been developed and publically available. It is an online open-access database for genome biology of ictalurid catfish (Ictalurus spp.). It serves as a comprehensive, integrative platform for all aspects of catfish genetics, genomics and related data resources. cBARBEL provides BLAST-based, fuzzy, and specific search functions, visualization of catfish linkage, physical, and integrated maps, a catfish EST contig viewer with SNP information overlay, and GBrowse-based organization of catfish genomic data based on sequence similarity to the zebrafish chromosomes. Subsections of the database are tightly-related, allowing a user with a sequence or search string of interest to navigate seamlessly from one area to another. As catfish genome sequencing proceeds and ongoing quantitative trait loci (QTL) projects bear fruit, cBARBEL will allow rapid data integration and dissemination within the catfish research community and to interested stakeholders. cBARBEL can be accessed at http://catfishgenome.org.
Publications
- Lu J, Peatman E, Wang W, Yang Q, Abernathy J, Wang S, Kucuktas H, Liu ZJ. 2010. Alternative splicing in teleost fish genomes: Same-species and cross-species analysis and comparisons. Molecular Genetics and Genomics 283:531-539.
- Liu ZJ. 2010. Development of genomic resources in support of sequencing, assembly, and annotation of the catfish genome. Comparative Biochemistry and Physiology, Part D, Genomics and Proteomics 6:11-17.
- Liu ZJ, Peatman E. 2010. Whole Genome Sequencing of Aquaculture Species: Identifying the Genotypes behind Production Phenotypes. Global Aquaculture Advocate 2010:68-70.
- Liu H, Takano T, Peatman E, Abernathy J, Wang S, Sha Z, Kucuktas H, Liu ZJ. 2010. Molecular characterization and gene expression of the channel catfish ferritin H subunit after bacterial infection and iron treatment. Journal of Experimental Zoology, Part A: Ecological Genetics and Physiology 313:359-368.
- Jiang Y, Abernathy J, Peatman E, Liu H, Wang S, Xu D-H, Kucuktas H, Klesius P, Liu Z.J. 2010. Identification and characterization of matrix metalloproteinase-13 sequence structure and expression during embryogenesis and infection in channel catfish (Ictalurus punctatus). Developmental and Comparative Immunology 34:590-597.
- Liu H, Tomokazu T, Abernathy J, Wang S, Sha Z, Terhune J, Kucuktas H, Jiang Y, Liu ZJ. 2010. Structure and expression of transferrin gene of channel catfish, Ictalurus punctatus. Fish and Shellfish Immunology 28:159-166.
- Lu J, Peatman E, Yang Q, Wang S, Hu Z, Kucuktas H, Liu ZJ. 2010. Catfish Genome Database cBARBEL: A informatic platform for genome biology of ictalurid catfish. Nucleic Acids Research 2010:1-7.
- Xu DH, Klesius P, Peatman E, and Liu ZJ. 2010. Susceptibility of channel catfish, blue catfish and channel and blue catfish hybrid to Ichthyophthirius multifiliis. Aquaculture 311:25-30.
- Chen F, Lee Y, Jiang Y, Wang S, Peatman E, Abernathy J, Liu H, Liu S, Kucuktas H, Ke C, Liu ZJ. 2010. Identification and characterization of full-length cDNAs in catfish (Ictalurus spp.). PLoS One 12:11546.
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