ena-STUDY-QMUL-14-11-2013-17:13:50:698-37
Genome sequence of Fraxinus excelsior (ash) with resequence data for 37 ash trees from across Europe and RNA-seq from four tissues. Plus whole genome shotgun bisulphite sequence data for Fraxinus excelsior and F. mandschurica.
This project sequenced the genome of a British Ash Tree. The tree being sequenced is the result of self-pollination by a tree growing in woodland in the Cotswolds. The controlled self-pollination of the parent tree was carried out by Dr David Boshier of Oxford University. The offspring from this self-pollination are growing at Paradise Wood in Oxfordshire, owned by the Earth Trust, and managed by Jo Clark. Tissue was collected from one of these trees in January 2013 and DNA was extracted from it by Jasmin Zohren at QMUL. Using flow cytometry, we estimated the 1C genome size of the tree to be 877 Mbp. Raw DNA sequence data for the British ash genome was generated by Eurofins, and the data was assembled by Lizzy Sollars and Richard Buggs at QMUL, in collaboration with CLCbio, using open access and proprietary software. Assembly and analysis of the genome was carried out on the QMUL-High Performance Computing MidPlus cluster, and servers at CLCbio. Reads from paired libraries of insert sizes: 200bp, 300bp, 500bp, and Long-Jumping Distance (LJD) libraries of 3kb, 8kb, 20kb and 40kb, were trimmed to a minimum Phred quality score of 20, a minimum length of 50bp, and were also trimmed of any adaptor and repetitive telomere sequences. Reads were assembled de novo using CLC bio with a word size (k-mer) of 50, into contigs with a minimum length of 500bp. The LJD pairs and 454 contigs assembled using Newbler (BATG-0.1-Newbler) were used as guidance reads to resolve ambiguities in the de bruijn graphs. Contigs were then scaffolded using the stand-alone tool SSPACE, and gaps in the scaffolds were closed using the GapCloser program from SOAP.   We also did RNA-SEQ from five tissues (four from the mother tree and one from the focal tree) using Illumina HiSeq.   A further 37 trees were sequenced by The Genome Analysis Centre in Norwich, UK, to an average of 8.4X genome coverage of trimmed and filtered reads per tree. These trees are from the European range of F. excelsior, growing in the "Realising Ash's Potential" provenance trial at Earth Trust, UK.  In addition (experiment accessions ERX2162802 to ERX2162821) we sequenced the methylomes of twenty ash trees using whole genome bisulphite sequencing (WGBS). These trees consisted of two grafted replicates of Danish Fraxinus excelsior Clone 27, and five grafted replicates each of Danish F. excelsior Clones 33, 35 and 40, two grafted replicates of one F. mandshurica genotype, one sample of another F. mandshurica genotype. Clones 33 and 35 have previously been shown to have consistent low susceptibility to ADB in field trials in Denmark and F. mandshurica is a species with low susceptibility to ADB. DNA was extracted from dried leaves of the twenty samples in December 2013 at the University of Copenhagen, using Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and checked for quality and quantity using Nanodrop (Thermo Scientific, Waltham, MA, USA). DNA samples were sent to the Genome Centre at QMUL where they were quantified using Qubit (Invitrogen Carlsbad, CA, United States) and vacuum concentrated. Between 200ng and 500ng of DNA from each sample were bisulphite-converted at the Genome Centre using the EZ DNA Methylation Gold kit (Zymo Research, Irvine, CA, USA). Libraries were prepared using EpiGnome Methyl-seq kit (Epicentre, Madison, WI, USA), and checked on an Agilent Tape Station using the DNA1000 High Sensitivity Screen Tape assay (Agilent, Santa Clara, CA, USA) and sequencing was carried out on an Illumina HiSeq 2500 instrument (Illumina, San Diego, USA), using 2 x 100 bp paired reads. After bisulphite conversion and sequencing, a total of 2,052 million raw reads were generated, with an average of 102.6 million reads (12x coverage) per sample.
This project sequenced the genome of a British Ash Tree. The tree being sequenced is the result of self-pollination by a tree growing in woodland in the Cotswolds. The controlled self-pollination of the parent tree was carried out by Dr David Boshier of Oxford University. The offspring from this self-pollination are growing at Paradise Wood in Oxfordshire, owned by the Earth Trust, and managed by Jo Clark. Tissue was collected from one of these trees in January 2013 and DNA was extracted from it by Jasmin Zohren at QMUL. Using flow cytometry, we estimated the 1C genome size of the tree to be 877 Mbp. Raw DNA sequence data for the British ash genome was generated by Eurofins, and the data was assembled by Lizzy Sollars and Richard Buggs at QMUL, in collaboration with CLCbio, using open access and proprietary software. Assembly and analysis of the genome was carried out on the QMUL-High Performance Computing MidPlus cluster, and servers at CLCbio. Reads from paired libraries of insert sizes: 200bp, 300bp, 500bp, and Long-Jumping Distance (LJD) libraries of 3kb, 8kb, 20kb and 40kb, were trimmed to a minimum Phred quality score of 20, a minimum length of 50bp, and were also trimmed of any adaptor and repetitive telomere sequences. Reads were assembled de novo using CLC bio with a word size (k-mer) of 50, into contigs with a minimum length of 500bp. The LJD pairs and 454 contigs assembled using Newbler (BATG-0.1-Newbler) were used as guidance reads to resolve ambiguities in the de bruijn graphs. Contigs were then scaffolded using the stand-alone tool SSPACE, and gaps in the scaffolds were closed using the GapCloser program from SOAP.   We also did RNA-SEQ from five tissues (four from the mother tree and one from the focal tree) using Illumina HiSeq.   A further 37 trees were sequenced by The Genome Analysis Centre in Norwich, UK, to an average of 8.4X genome coverage of trimmed and filtered reads per tree. These trees are from the European range of F. excelsior, growing in the "Realising Ash's Potential" provenance trial at Earth Trust, UK.  In addition (experiment accessions ERX2162802 to ERX2162821) we sequenced the methylomes of twenty ash trees using whole genome bisulphite sequencing (WGBS). These trees consisted of two grafted replicates of Danish Fraxinus excelsior Clone 27, and five grafted replicates each of Danish F. excelsior Clones 33, 35 and 40, two grafted replicates of one F. mandshurica genotype, one sample of another F. mandshurica genotype. Clones 33 and 35 have previously been shown to have consistent low susceptibility to ADB in field trials in Denmark and F. mandshurica is a species with low susceptibility to ADB. DNA was extracted from dried leaves of the twenty samples in December 2013 at the University of Copenhagen, using Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and checked for quality and quantity using Nanodrop (Thermo Scientific, Waltham, MA, USA). DNA samples were sent to the Genome Centre at QMUL where they were quantified using Qubit (Invitrogen Carlsbad, CA, United States) and vacuum concentrated. Between 200ng and 500ng of DNA from each sample were bisulphite-converted at the Genome Centre using the EZ DNA Methylation Gold kit (Zymo Research, Irvine, CA, USA). Libraries were prepared using EpiGnome Methyl-seq kit (Epicentre, Madison, WI, USA), and checked on an Agilent Tape Station using the DNA1000 High Sensitivity Screen Tape assay (Agilent, Santa Clara, CA, USA) and sequencing was carried out on an Illumina HiSeq 2500 instrument (Illumina, San Diego, USA), using 2 x 100 bp paired reads. After bisulphite conversion and sequencing, a total of 2,052 million raw reads were generated, with an average of 102.6 million reads (12x coverage) per sample.