#ITIS:(Identify Transposon Insertion Sites)
is a pipeline to identify novel TE insertion sites in genome
It require three input files:
(i) reference genome sequence,
(ii)TE sequence,
(iii)paired-end (PE) short reads, with no restriction on length,generated from the re-sequenced genome that contains novel TE insertions.
By aligning read pairs to merged reference sequence, reference genome and TE sequence, ITIS will check each informative read pairs as long as it have more than 20bp overlap with TE sequence and determine if it supports the TE insertion around the location mapped by one of read pair. In theory, by inspecting both cross read pairs and clipped reads at the same time, ITIS will have a higher sensitivity than other tools
NOTE: ITIS should be used to identy de novo insertions sites, It is unable to detect the lose event of one preexisting TEs.
##Table of Contents
The following programs need to be installed and the executable commands should be in $PATH of system:
samtools (v 0.1.19) #####******IMPORTANT******######
bwa (v 0.7.7-r441)
bedtools (v 2.17.0)
Bio::Perl
blast+
R
Other usefull tool:
IGV
USAGE:
/psc/home/jiangchuan/Dropbox/Code/Code_TE_inser/itis.pl
REQUIRED -g the genome sequence file in fasta format
OR -G prefix of bwa-indexed reference file ( genome + transposon)
REQUIRED -t the TE sequence file in fasta format
or -T prefix of bwa-indexed transposon sequence file
REQUIRED -l the average length of fragments in library
REQUIRED -N the name of project
REQUIRED -1 the paired read file 1
REQUIRED -2 the paired read file 2
-f <gff file> if provided, ITIS will check if TE inserted in gentic or intergeneic region
-F <Y|N> run scripts in 'FAST' mode; It won't align all reads to reference genome,caculate the average bg depth,
and estimate if insertion is homo or heter,[default N]
## parameters used with '-F N' :
-B <bam file> use previous sorted and indexed bam file of reads aligned to reference genome
-d <Num,Num> the depth range to filter raw insertion site, [default 2,200]
-q <Num> the minimum average mapping quality of all supporting reads, [default 1]
-e <Y|N> If reference genome contains this TE or it's homolog. using blast to hard mask these sequence is required, [default N]
-a <Num> the number of bases allowed to be lost when transposing, [defualt 10]
-b <tags> minimum required number of flanking reads , in the format of /Tag=Value/Tag=Value/Tag=Value/ , the avaliable tags:
t: total supporting reads at detected insertion /t=3/
CS:clipped reads cover TE start site /CS=0/
CE:clipped reads cover TE end site /CE=0/
cs:cross reads cover TE start /cs=0/
ce:cross reads cover TE end /cs=0/
TS:total reads cover TE start /TS=1/
TE:total reads cover TE end /TE=1/
[default /t=3/TS=1/TE=1/]
-c <Num,Num,Num> cpu number for 'BWA mem', 'samtools view' and 'samtools sort', [defualt 8,2,2]
-w <Num> window size used to cluster supportting reads, [default library_length/2]
-D <Directory> use this specifed temperate directory, [default[project].[aStringOfNumbers]]
-m <Y|N> Only print out all commands to STDERR, [default N]
-h print this help message
eg: perl /psc/home/jiangchuan/Dropbox/Code/Code_TE_inser/itis.pl -g genome.fa -t tnt1.fa -l 300 -N test_run -1 reads.fq1 -2 reads.fq2 -f medicago.gff3
this test dataset derived from the genome resequencing project of Japonica A123(SRR631734), which have transposon mping be activted.
All PE reads mapped at chr1:1-2000000 were extracted and saved in file sample.fq1 and sample.fq2.
First of all, untar the sample dataset:
cd test_dir
tar xvzf sample_data.tar.gz
####Input Files
PE reads:
sample.fq1
sample.fq1
reference genome:
rice_chr1_200k.fa
Transposon sequence:
mping.fa
####command to detect mping insertions in reference genome perl path/to/itis.pl -g rice_chr1_200k.fa -t mping.fa -l 500 -N test -1 sample.fq1 -2 sample.fq2 -e Y
\#-e Y : to tell itis.pl that there are mping homologous sequence in reference genome
####Output Files itis will produce a lot of files in a directory named test.[aStringOfNumbers]
The important files included:
test.mping.filtered.bed
This is a list of reliable insertion sites.
TAGS in column 4:
SR=(NO. of supporting library fragment),(No. supporting reads),(No. clipped reads at TE start),(No. clipped reads at TE end),(No. cross reads at TE start),(No. cross reads at TE end)
MQ=average mapping quality of all supporting reads
NM=name of TE
GT=(No. of supporting reads),(No. of background reads):(Heter|Homo)
PV=the p-value ofbinomial test for zygosity based on the GT values
DP=average depth of 100bp region flanking the TE insertion site
TS=the joint position at the begin of TE
TE=the joint pisition at the end of TE
result contents with test_data:
Chr1:0-2000000 174497 174500 SR=8,9,6,2,0,1;MQ=57;NM=mping;GT=8,0:Heter;PV=0.03125;DP=16;TS=1;TE=430;NB=N . +
Chr1:0-2000000 214352 214355 SR=15,17,9,5,1,2;MQ=57;NM=mping;GT=15,0:Homo;PV=0.00390625;DP=17;TS=1;TE=430;NB=N . +
Chr1:0-2000000 316534 316537 SR=9,14,3,11,0,0;MQ=57;NM=mping;GT=9,0:Heter;PV=0.03125;DP=20;TS=1;TE=430;NB=N . -
Chr1:0-2000000 639972 639975 SR=8,8,4,3,0,1;MQ=51;NM=mping;GT=8,14:Heter;PV=0.9903946;DP=28;TS=1;TE=430;NB=N . +
Chr1:0-2000000 1193504 1193507 SR=9,12,5,7,0,0;MQ=56;NM=mping;GT=9,0:Heter;PV=0.03125;DP=18;TS=1;TE=430;NB=N . +
Chr1:0-2000000 1374936 1374939 SR=8,10,7,2,0,1;MQ=59;NM=mping;GT=8,10:Heter;PV=0.9407654;DP=28;TS=1;TE=430;NB=N . +
test.mping.raw.bed
This is all candidate insertion sites, some of which may be false
test.mping.support.reads.sam and test.mping.support.reads.sorted.bam
This is alignment file of all supportive reads
test.all_reads_aln_ref_and_te.sort.bam
This is alignment file of all reads
commands_rcd
A record of all the command used by itis.pl to identify TE insertions.
test.ref_and_te.fa
the reference sequence, containing genome and mping sequence, used by bwa to align reads.
*bam, *bed and reference sequence can be visuallized in IGV
If you want to filter the raw insertion list by personalized criteria, you can rerun the script filter_insertion.pl, just as shown in command_rcd
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