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| Status |
Public on May 01, 2014 |
| Title |
TBS-seq for 1ng repeat2 |
| Sample type |
SRA |
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| Source name |
rice leaf
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| Organism |
Oryza sativa |
| Characteristics |
tissue: leaf
input(ng): 1
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA prepared from a rice strain was quantified by QuantiT dsDNA HS assay kit (0.2-100ng; Invitrogen, Q32854, Carlsbad, CA, USA).
10ng ( each aliquot was spiked with 10pg of unmethylated lambda DNA), 1ng and 0.5ng aliquots of genomic DNA were prepared by gradient dilution and then subjected to fragmentation reaction (regardless of the quantity of DNA used ) by incubating with 4 µL Nextera HMW Buffer (Epicentre-Illumina), nuclease-free water (Ambion) to 16 uL and 4 µL prepared Tn5mC transposomes for 12 min at 55°C followed by purification using 36 µl (1.8X) Agencourt AMPure XP magnetic beads with elution in 14 µL EB buffer. An extension step was then performed by adding 2 µL of 10X Thermopol reation buffer (NEB), 2 µL 10mM 5-Methylcytosine dNTP Mix (Zymo Research, D1030), 1 µl of Bst DNA Polymerase Large Fragment (NEB, M0275s) to each reaction and incubating for 20 min at 65°C. Each reaction was spiked with 200ng of sonicated unmethylated lambda DNA (200-400 bp) and then subjected to bisulfite conversion using a Zymo EZ DNA Methylation Kit (D5100) according to recommended protocols with a 14h 50°C dark incubation and 22 µL (2 x11 µL) elution. The purified DNAs were then amplified using 25 µL Kapa 2G Robust Hot Start Ready Mix (Kapa Biosystems), 1 µL 50X Nextera Primer Cocktail (Illumina - compatible) and 1 µL barcoded Illumina-compatible Adaptor 2 (8-bp index) on the thermocycler with the following parameters: 1 min at 95°C; (20 sec at 95°C; 30 sec at 60°C; 45 sec at 72°C) X (10-15) cycles. The prepared libraries were analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by qPCR and then used for cluster generation and pair-end sequencing with 90 bp reads (PE90) on Illumina Hiseq 2000 using the following sequencing primers: read 1, 5’-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGA-3’; read 2, 5’-GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGA-3; and index read, 5’-TCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG-3. We generated two chemistry ligation based WGBS libraries from 1000 ng of genomic DNA prepared from Rice (each library was spiked with 1ng of unmethylated lambda DNA) according to methods described previously [10]. Libraries were sequenced in a strategy with pair-end 90 bp read (PE90) on Illumina Hiseq 2000 according to the standard Illumina protocol.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
tagmentation-based whole genome bisulfite sequencing
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| Data processing |
After sequencing, adapters were first removed using cutadapt-1.2.1 software (http://code.google.com/p/cutadapt/), only reads with minimally 45 bp length were remained. After removing adapters, low quality reads that contained more than 10% N’s or more than 50% of the sequence with low quality value (quality value <5) per read were filtered out. Read mapping was done on the rice reference genome (IRGSP_build5) using BS-MAP (version 2.73) alignment software that combines genome hashing and bitwise masking to achieve fast and accurate bisulfite mapping, allowing for maximally 5 mismatches or 1 small insertion or deletion.
After mapping, PCR duplicates were removed using Samtools software. Then, we separated the reads into two categories, depending on whether they are from the top (C changed to T, C-T) and bottom (G changed to A, G-A) strands. By simulating the methylation proportion across each possible read position, the 12 bps from the 3’ ends of C-T reads and the 5’ ends of G-A reads were then removed as which were affected by the 5-methyl-dCTPs incorporated during the gap-repair and strand displacement step.
Methylation level was defined as the fraction of read counts of ‘C’ in the total read counts of both ‘C’ and ‘T’ for each covered C site, and herein average percentage methylation of all cytosine residues for any genomic region covered was computed as the fraction of read counts of ‘C’ in the total read counts of both ‘C’ and ‘T’ for each genomic region. Based on such read fraction, methylated cytosines were called using a binomial distribution as in the method described by Lister et al. (2009), whereby a probability mass function is calculated for each methylation context (CpG, CHG, CHH). The non-conversion rate and sequencing error rate were taken into account, for which the total error rate was determined by unmethylated lambda DNA spike-ins
We comprehensively assessed the repeatability, sensitivity as well as specificity and characterized both global and genic methylation patterns of a rice strain by this new T-WGBS, proving its consistency in comparison with LC-WGBS.
Genome_build: IRGSP_build5
Supplementary_files_format_and_content: *.cout.gz files contain 8 columns. The format is that: (1)Chromosome ID, (2)Cytosine position, (3)+/- Watson/Crick chain, (4)Type (CG/CHH/CHG), (5)Base pattern, (6)Methylation Ratio, (7)Methylated reads num, (8)Un-methylated reads num.
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| Submission date |
Apr 24, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Desheng Gong |
| E-mail(s) |
gds19870718@163.com
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| Organization name |
Agricultural Genomes Institute at Shenzhen
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| Street address |
No.7 PengFei road
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| City |
Shenzhen |
| ZIP/Postal code |
518120 |
| Country |
China |
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| Platform ID |
GPL13160 |
| Series (1) |
| GSE57062 |
Improved tagmentation-based whole genome bisulfite sequencing for input DNA from less than 100 cells |
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| Relations |
| BioSample |
SAMN02736980 |
| SRA |
SRX526690 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1373867_tbs_1ng_r2.cout.txt.gz |
803.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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