Dinh 2011/NOTES/2011-12-7

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DIP-seq for 5caC and 5fC in mouse ESC[edit]

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]

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]

 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]