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Browse wiki
From NematodeGenomes
| Has interested party
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Richard Davis +,
Matt Berriman +,
Mark Blaxter +,
Robin Gasser +,
Aaron Jex +
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| Modification dateThis property is a special property in this wiki.
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8 November 2011 12:31:45 +
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| Strain bclade
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Bclade III +
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| Strain genome contact
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Robin Gasser +,
Aaron Jex +
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| Strain genome funder
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Australian Research Council +
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| Strain genome plan
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DNA isolation, sequencing and quality cont … DNA isolation, sequencing and quality control
Total genomic DNA was isolated from the reproductive tract of a single adult female of A. suum using a sodium-dodecyl sulphate/proteinase K digestion33 followed by phenol-chloroform extraction and ethanol precipitation34. Total DNA yield was determined using the Qubit fluorometer double-stranded DNA HS Kit (Invitrogen). DNA integrity was verified with a 2100 Bioanalyser (Agilent). Short-insert (170 bp and 500 bp) and mate-pair (800 bp, 2 kb, 5 kb and 10 kb) genomic DNA libraries were prepared and paired-end sequenced using TruSeq chemistry on a HiSeq 2000 (Illumina). Whole-genome amplification, employing the REPLI-g Midi Kit (Qiagen), was used to produce (from 200 ng of genomic template) the required amount of DNA for the construction of the 2-kb, 5-kb and 10-kb libraries (Supplementary Fig. 6). The sequence data generated from each of the six libraries were verified, and low-quality sequences, base-calling duplicates and adapters removed. The size of the genome and the heterozygosity rate were estimated by establishing the frequency of occurrence of each 17-bp k-mer (a unique sequence of k (that is, 17) nucleotides in length) within the genomic sequence data set (from the 170-bp library) using an established method5. Genome size was estimated using a modification of the Lander–Waterman algorithm35, where the haploid genome length in base pairs is G = (N × (L − K + 1) − B)/D, where N is the read length sequenced in base pairs, L is the mean length of sequence reads, K is the k-mer length (17 bp) and B is the number of k-mers occurring less than four times (Supplementary Fig. 7). Heterozygosity was evaluated throughout the genome assembly by assessing the distribution of the k-mer frequency in the sequence data set. k-mer frequency in the sequence data set.
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| Strain genome status
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published +
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| Strain genome url
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ftp://ftp.wormbase.org/pub/wormbase/species/a_suum/ +
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| Strain is reference
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false +
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| Strain mitochondrion status
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none +
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| Strain symbiont status
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none +
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| Strain transcriptome contact
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Robin Gasser +,
Aaron Jex +
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| Strain transcriptome funder
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Australian Research Council +
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| Strain transcriptome plan
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RNA isolation, sequencing and assembly
We … RNA isolation, sequencing and assembly
We obtained total RNAs from egg-L3s (n ≈ 500,000), liver-L3s (n ≈ 60,000), lung-L3s (n ≈ 80,000) or L4s (n ≈ 30,000) and from the somatic musculature or reproductive tract of each of two adult male and two adult female A. suum using the TriPure reagent (Roche), and both yield and quality were verified by 2100 BioAnalyser (Agilent). Polyadenylated (polyA+) RNA was purified from 10 μg of total RNA using Sera-mag oligo(dT) beads, fragmented to a size of 300–500 bp, reverse-transcribed using random hexamers, end-repaired and adaptor-ligated, according to the manufacturer’s protocol (Illumina). Ligated products of ~400 bp were excised from agarose and then PCR-amplified (15 cycles), as recommended. Products were purified over a MinElute column (Qiagen) and subjected to paired-end RNA-seq using TruSeq chemistry on a HiSeq 2000 (Illumina) and assessed for quality and adaptor sequence. Transcripts were assembled from RNA-seq data using Oases36. All transcripts were used to assess the completeness of the genome assembly and to predict genes. the genome assembly and to predict genes.
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| Strain transcriptome status
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published +
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| Taxon parent
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Ascaris suum +
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| Categories |
Strain
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