Dinh 2011/NOTES/2011-12-7
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DIP-seq for 5caC and 5fC in mouse ESC[edit]
- Followup from http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh_2011/NOTES/2011-11-22
- After removing other rodent repeats and keeping only the mouse repeats, I blasted the hits again to the repeat database (keeping the best hit for each multiple mapping reads)
- I need to make these numbers into pie-charts.
Repeat type | Input | IgG | 5caC | 5fC |
C573 | 0 | 0 | 1 | 0 |
Mariner/Tc1 | 1 | 2 | 0 | 0 |
Repeat | 2 | 0 | 0 | 0 |
Simple | 2 | 0 | 0 | 0 |
MCMREP | 4 | 8 | 0 | 0 |
RMER1A | 4 | 1 | 0 | 0 |
Nonautonomous | 5 | 3 | 0 | 1 |
RMER1 | 5 | 1 | 0 | 2 |
RMER1B | 5 | 6 | 0 | 0 |
PMR | 7 | 0 | 1 | 0 |
Y | 15 | 10 | 6 | 5 |
hAT | 27 | 14 | 9 | 2 |
DNA | 57 | 60 | 75 | 19 |
Internal | 93 | 61 | 55 | 18 |
Transposable | 106 | 59 | 0 | 25 |
Satellite | 170 | 129 | 246 | 58 |
Endogenous | 416 | 346 | 227 | 85 |
SINE | 676 | 358 | 478 | 159 |
LTR | 686 | 490 | 859 | 209 |
ERV1 | 1174 | 944 | 983 | 404 |
SINE2/tRNA | 2532 | 1236 | 1148 | 443 |
SINE1/7SL | 2649 | 1520 | 2007 | 693 |
ERV3 | 5596 | 3435 | 1927 | 912 |
ERV2 | 7762 | 5921 | 5909 | 2027 |
L1 | 32292 | 28783 | 24410 | 9819 |
SAT | 45714 | 56613 | 61659 | 85119 |
- 5caC and 5fC are definitely enriched at SATELLITE repeat elements.
- Here are Dr. Zhang's repeat analysis
Data_file | Libraries | % unique mappable reads | % non-unique mappable reads | # mapped to rodent repeats* | # mapped to GSAT* | % GSAT repeats | # mapped to L1* | % L1 |
s_1 | YZ_UNC-5caC-DIP-Oct27 | 53.6% | 40.7% | 450 | 200 | 44.4% | 8900.0% | 19.8% |
s_2 | YZ_UNC-5caC-DIP-Oct27 | 53.1% | 41.4% | 427 | 170 | 39.8% | 8300.0% | 19.4% |
s_5 | YZ_UNC-5fC-DIP-Oct27 | 44.0% | 44.6% | 709 | 643 | 90.7% | 2800.0% | 3.9% |
s_4 | YZ_UNC-IgG-DIP-Oct27 | 64.1% | 27.3% | 459 | 284 | 61.9% | 9500.0% | 20.7% |
s_3 | YZ_UNC-INPUT-Oct27 | 65.5% | 19.7% | 582 | 256 | 44.0% | 16000.0% | 27.5% |
Paper:[edit]
- Aberrant Overexpression of Satellite Repeats in Pancreatic and Other Epithelial Cancers, http://www.sciencemag.org/content/331/6017/593.full
- Some notes from the supplementary on gene expression and satellite expression profiling
1) Reads were aligned to a custom made library for the mouse major satellite sequence from UCSC genome browsere 2) Reads aligning to the repeat reference were counted and their fraction of the total number of genome aligned reads was determined 3) Gene expression counts were determined based on alignments to the annotated transcriptomes based on a statistical gene counting method 4) Gene counts were normalized per million aligned genomic reads for all samples 5) For linear correlation of mouse major satellite to transcriptome, linear regression analysis of the normalized expression levels of each gene across all tissues vs the measured satellite expression level. 6) Genes were then ranked according to decreasing Pearson coefficient and a set of highly correlating genes (R>0.85) was selected for further analysis.
SOAP3 (latest version)[edit]
- Downloaded and install the latest binary release of SOAP3 from http://www.cs.hku.hk/2bwt-tools/soap3/
genemapster: /media/2TB_store2/Dinh_Scratch/softwares/soap3-r146
- Add to path:
export PATH=/media/2TB_store2/Dinh_Scratch/softwares/soap3-r146:$PATH
- Index reference hg18
In directory /media/2TB_store2/Dinh_Scratch/HsRef/Hg18/ soap3-builder genome.fa
- Convert to GPU Index
BGS-Build genome.fa.index
- Edit soap3_aligner.ini file:
SaValueFileExt=.sa NumOfCpuThreads=4 HostAlignmentModel=16G MaxOutputPerRead=1 StrandArrangement=+/- MaxOutputPerPair=1 GPUMemory=4
- Ran soap3_aligner (2 mismatch, unique best alignments only):
soap3_aligner single /media/2TB_store2/Dinh_Scratch/HsRef/Hg18/HsGenome18.fa.index test1000000reads.fastq -m 2 -h 3
- Outputs:
[Main] Finished parsing ini file soap3_aligner.ini. [Main] Finished loading index into host. [Main] Loading time : 3.0066 seconds [Main] Reference sequence length : 2858029421 [Main] Finished copying index into device (GPU). [Main] Loading time : 0.8665 seconds [Main] Loaded 1000000 short reads from the query file. [Main] Elapsed time on host : 1.3785 seconds Finished alignment with <= 2 mismatches Elapsed time : 0.1998 seconds Number of reads aligned: 1000000 (number of alignments: 1000000) [Main] Overall number of reads aligned: 1000000 (number of alignments: 1000000) [Main] Overall number of unaligned reads: 0 [Main] Overall read load time : 1.3785 seconds [Main] Overall alignment time (excl. read loading) : 0.1998 seconds [Main] Free device memory.. [Main] Free index from host memory.. [Main] Free host memory.. [Main] soap3_aligner terminated. Please type the following command to merge the alignment results into one file: ./make_view_simple.sh test1000000reads.fastq
- Run make_view_simple.sh
make_view_simple.sh test1000000reads.fastq
- Outputs:
------------------------------------------------------------- Alignment file - test1000000reads.fastq.out has been created Format: Read #, Chromosome Id#, Offset, Strand, # of Mismatch -------------------------------------------------------------
- head -5 test1000000reads.fastq.out
1 1 50000 + 0 2 1 50000 + 0 3 1 50000 + 0 4 1 50000 + 0 5 1 50000 + 0
- Alignments are all wrong
SOAP3 try compiling from source[edit]
- Downloaded http://www.cs.hku.hk/2bwt-tools/downloads/soap3-r146-source.tar.gz
- Still didn't work
- SOAP3 requires a GPU with compute capability of 2.0 (Tesla C1060 is of 1.3)
- SOAP3 website only mentioned that they require CUDA Driver version 3.2...they should probably also state the compute capability requirement.