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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Aug 28, 2013 |
| Title |
BC05 PB3 |
| Sample type |
SRA |
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| Source name |
tumor, intestine
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| Organism |
Mus musculus |
| Characteristics |
strain/background: mixed 129/B6/Swiss
tissue: intestine tumor
sample: tumor 11
transposon side: PB3
barcode: ACATGT
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| Treatment protocol |
Tumors were obtained in mice between 59-83 weeks of age.
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| Growth protocol |
Tumors were obtained in mice that carried a constitutive expressed PiggyBac transposase allele (ROSA26-PBase) and ATP1 transposon array (ATP1-S2).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from tumors and tail controls by standard DNA extraction methods with SDS lysis buffer and proteinase K digestion.
DNA was fragmented by acoustic shearing, followed by DNA end repair, A-tailing of 3'-ends, and ligation to Splinkerette adapters with 3'-T-overhang. In separate reactions, junction fragments for the 3' and 5' ends of the PiggyBac transposon (PB3 and PB5) were then amplified in two consecutive PCR rounds to generate PB3 and PB5 libraries for each sample. PCR primers contained terminal adapter sequences for Illumina solid-phase amplification and sequencing and a 6-base barcode to distinguish samples in multiplex sequencing (BC01:ATCACG, BC02:CGATGT, BC03:TTAGGC, BC04:TGACCA, BC05:ACATGT, BC06:GCCAAT, BC07:CAGATC, BC08:ACTTGA, BC09:GATCAG, BC10:TAGCTT, BC11:GGCTAG, BC12:CTTGTA, BC13:CGTGAT, BC14:ACATCG, BC15:GCCTAA, BC16:TGGTCA, BC17:CACTGT). Two pools of PB3 and PB5 libraries were sequenced on single lanes of an Illumina HiSeq 2000 device with 100 bases read length.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
After demultiplexing of barcoded reads, PiggyBac transposon sequences were trimmed.
Remaining sequences were aligned to the mouse genome (GRCm38/mm10) using the Bowtie algorithm, with a seed size of 20 nucleotides, 1 allowed mismatch in the seed and any number of mismatches in the remaining read. Only the best alignment for each read was retained.
In a second step, reads were trimmed so that the resulting alignment contained at most 4 mismatches.
Reads that contained a starting position within a gamma satellite repeat element were filtered out, using annotations by RepeatMasker (www.repeatmasker.org).
Subsequently, reads were clustered according to their starting positions (corresponding to transposon insertion sites), and only starting positions and corresponding reads were retained that were supported by at least one read of a minimum size of n bases. To determine n, we used the fraction of reads from insertions that were supported by reads on both PB3 and PB5 sides as a guideline for assessing the quality of our sequence mappings. A minimum read length of n=34 was chosen for further analysis as it was the smallest cutoff value that provided for almost all samples high quality sequence mapping, while higher values of n provided no significant gain in mapping quality but lead to a decrease in the number of identified insertion sites.
Genome_build: GRCm38
Supplementary_files_format_and_content: bedGraph files were created using BEDTools version 2.16.2.
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| Submission date |
Apr 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Caroline Friedel |
| Organization name |
Ludwig-Maximilians-Universität München
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| Department |
Institut für Informatik
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| Street address |
Amalienstr. 17
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| City |
München |
| ZIP/Postal code |
80333 |
| Country |
Germany |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE46210 |
Clonal expansion analysis of transposon insertions by high-throughput sequencing identifies candidate cancer genes in a PiggyBac mutagenesis screen |
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| Relations |
| BioSample |
SAMN02052411 |
| SRA |
SRX268564 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1126401_PB3_BC05_mm1_34_repeats_filtered.bedgraph.gz |
215.7 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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