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With the improvement of sequencing techniques, chromatinimmunoprecipitation followed by high throughput sequencing (ChIP-Seq)is getting popular to study genome-wide protein-DNA interactions. Toaddress the lack of powerful ChIP-Seq analysis method, we present anovel algorithm, named Model-based Analysis of ChIP-Seq (MACS), foridentifying transcript factor binding sites. MACS captures theinfluence of genome complexity to evaluate the significance ofenriched ChIP regions, and MACS improves the spatial resolution ofbinding sites through combining the information of both sequencing tagposition and orientation. MACS can be easily used for ChIP-Seq dataalone, or with control sample with the increase of specificity.
Install
Please check the file 'INSTALL' in the distribution.
![]() Usage
Example for regular peak calling:
macs2 callpeak -t ChIP.bam -c Control.bam -f BAM -g hs -n test -B -q 0.01
Windows 7 free download with product key. Example for broad peak calling:
macs2 callpeak -t ChIP.bam -c Control.bam --broad -g hs --broad-cutoff 0.1
There are seven major functions available in MACS serving as sub-commands.
We only cover 'callpeak' module in this document. Please use 'macs2COMMAND -h' to see the detail description for each option of eachmodule.
Call peaks
This is the main function in MACS2. It can be invoked by 'macs2callpeak' command. If you type this command without parameters, youwill see a full description of commandline options. Here we only listthe essential options.
Essential Options-t/--treatment FILENAME
This is the only REQUIRED parameter for MACS. File can be in anysupported format specified by --format option. Check --format fordetail. If you have more than one alignment files, you can specifythem as
-t A B C . MACS will pool up all these files together.
-c/--control
The control or mock data file. Please follow the same direction as for-t/--treatment.
-n/--name
The name string of the experiment. MACS will use this string NAME tocreate output files like
NAME_peaks.xls , NAME_negative_peaks.xls ,NAME_peaks.bed , NAME_summits.bed , NAME_model.r Download game stranded deep 64 bit. and so on. Soplease avoid any confliction between these filenames and yourexisting files.
--outdir
MACS2 will save all output files into speficied folder for thisoption.
-f/--format FORMAT
Format of tag file, can be 'ELAND', 'BED', 'ELANDMULTI','ELANDEXPORT', 'ELANDMULTIPET' (for pair-end tags), 'SAM', 'BAM','BOWTIE', 'BAMPE' or 'BEDPE'. Default is 'AUTO' which will allow MACSto decide the format automatically. 'AUTO' is also usefule when youcombine different formats of files. Note that MACS can't detect'BAMPE' or 'BEDPE' format with 'AUTO', and you have to implicitlyspecify the format for 'BAMPE' and 'BEDPE'.
Nowadays, the most common formats are BED or BAM/SAM.
BED
The BED format can be found at UCSC genome browser website.
The essential columns in BED format input are the 1st column'chromosome name', the 2nd 'start position', the 3rd 'end position',and the 6th, 'strand'.
BAM/SAM
If the format is BAM/SAM, please check the definition in(http://samtools.sourceforge.net/samtools.shtml). If the BAM file isgenerated for paired-end data, MACS will only keep the left mate(5'end) tag. However, when format BAMPE is specified, MACS will use thereal fragments inferred from alignment results for reads pileup.
BEDPE or BAMPE
A special mode will be triggered while format is specified as'BAMPE' or 'BEDPE'. In this way, MACS2 will process the BAM or BEDfiles as paired-end data. Instead of building bimodal distribution ofplus and minus strand reads to predict fragment size, MACS2 willuse actual insert sizes of pairs of reads to build fragmentpileup.
The BAMPE format is just BAM format containing paired-end alignmentinformation, such as those from BWA or BOWTIE.
The BEDPE format is a simplified and more flexible BED format, whichonly contains the first three columns defining the chromosome name,left and right position of the fragment from Paired-endsequencing. Please note, this is NOT the same format used by BEDTOOLS,and BEDTOOLS version of BEDPE is actually not in a standard BEDformat.
BOWTIE
If the format is BOWTIE, you need to provide the ASCII bowtie outputfile with the suffix '.map'. Please note that, you need to make surethat in the bowtie output, you only keep one location for oneread. Check the bowtie manual for detail if you want at(http://bowtie-bio.sourceforge.net/manual.shtml)
Here is the definition for Bowtie output in ASCII characters I copiedfrom the above webpage:
ELAND
If the format is ELAND, the file must be ELAND result output file,each line MUST represents only ONE tag, with fields of: https://evercss658.weebly.com/blog/watch-online-and-download-free-asian-drama.
ELANDMULTI
If the format is ELANDMULTI, the file must be ELAND output file frommultiple-match mode, each line MUST represents only ONE tag, withfields of:
Notes
-g/--gsize
PLEASE assign this parameter to fit your needs!
It's the mappable genome size or effective genome size which isdefined as the genome size which can be sequenced. Because of therepetitive features on the chromsomes, the actual mappable genome sizewill be smaller than the original size, about 90% or 70% of the genomesize. The default hs -- 2.7e9 is recommended for UCSC human hg18assembly. Here are all precompiled parameters for effective genomesize:
-s/--tsize
The size of sequencing tags. If you don't specify it, MACS will try touse the first 10 sequences from your input treatment file to determinethe tag size. Specifying it will override the automatically determinedtag size.
-q/--qvalue
The qvalue (minimum FDR) cutoff to call significant regions. Defaultis 0.05. For broad marks, you can try 0.05 as cutoff. Q-values arecalculated from p-values using Benjamini-Hochberg procedure.
Download Game Nong Tr I Vui V 2017-p/--pvalue
The pvalue cutoff. If -p is specified, MACS2 will use pvalue insteadof qvalue.
--nolambda
With this flag on, MACS will use the background lambda as locallambda. This means MACS will not consider the local bias at peakcandidate regions.
--slocal, --llocal
These two parameters control which two levels of regions will bechecked around the peak regions to calculate the maximum lambda aslocal lambda. By default, MACS considers 1000bp for small localregion(--slocal), and 10000bps for large local region(--llocal) whichcaptures the bias from a long range effect like an open chromatindomain. You can tweak these according to your project. Remember thatif the region is set too small, a sharp spike in the input data maykill the significant peak.
--nomodel
While on, MACS will bypass building the shifting model.
--extsizeDownload Game Nong Tr I Vui V 2 Online
While '--nomodel' is set, MACS uses this parameter to extend reads in5'->3' direction to fix-sized fragments. For example, if the size ofbinding region for your transcription factor is 200 bp, and you wantto bypass the model building by MACS, this parameter can be setas 200. This option is only valid when --nomodel is set or when MACSfails to build model and --fix-bimodal is on.
--shift
Note, this is NOT the legacy --shiftsize option which is replaced by--extsize! You can set an arbitrary shift in bp here. Please Usediscretion while setting it other than default value (0). When--nomodel is set, MACS will use this value to move cutting ends (5')then apply --extsize from 5' to 3' direction to extend them tofragments. When this value is negative, ends will be moved toward3'->5' direction, otherwise 5'->3' direction. Recommended to keep itas default 0 for ChIP-Seq datasets, or -1 * half of EXTSIZE togetherwith --extsize option for detecting enriched cutting loci such ascertain DNAseI-Seq datasets. Note, you can't set values other than 0if format is BAMPE or BEDPE for paired-end data. Default is 0.
Here are some examples for combining --shift and --extsize:
--keep-dup
It controls the MACS behavior towards duplicate tags at the exact samelocation -- the same coordination and the same strand. The default'auto' option makes MACS calculate the maximum tags at the exact samelocation based on binomal distribution using 1e-5 as pvalue cutoff;and the 'all' option keeps every tags. If an integer is given, atmost this number of tags will be kept at the same location. Empire earth 2 free download. Thedefault is to keep one tag at the same location. Default: 1
--broad
When this flag is on, MACS will try to composite broad regions inBED12 ( a gene-model-like format ) by putting nearby highly enrichedregions into a broad region with loose cutoff. The broad region iscontrolled by another cutoff through --broad-cutoff. The maximumlength of broad region length is 4 times of d from MACS. DEFAULT:False
--broad-cutoff
Cutoff for broad region. This option is not available unless --broadis set. If -p is set, this is a pvalue cutoff, otherwise, it's aqvalue cutoff. DEFAULT: 0.1
--scale-to <large|small>
When set to 'large', linearly scale the smaller dataset to the samedepth as larger dataset. By default or being set as 'small', thelarger dataset will be scaled towards the smaller dataset. Beware, toscale up small data would cause more false positives.
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If this flag is on, MACS will store the fragment pileup, controllambda, -log10pvalue and -log10qvalue scores in bedGraph files. ThebedGraph files will be stored in current directory named
NAME_treat_pileup.bdg for treatment data, NAME_control_lambda.bdg for local lambda values from control, NAME_treat_pvalue.bdg forPoisson pvalue scores (in -log10(pvalue) form), andNAME_treat_qvalue.bdg for q-value scores fromBenjamini–Hochberg–Yekutieli procedure.
--call-summits
MACS will now reanalyze the shape of signal profile (p or q-scoredepending on cutoff setting) to deconvolve subpeaks within each peakcalled from general procedure. It's highly recommended to detectadjacent binding events. While used, the output subpeaks of a bigpeak region will have the same peak boundaries, and different scoresand peak summit positions.
Output files
Other useful linksTips of fine-tuning peak callingDownload Game Nong Tr I Vui V 2 1
Check the three scripts within MACSv2 package:
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