===================================================================
README file for the AlleleDB pipeline v2.0
===================================================================
2016-04-18

CITATION:

[1] A uniform survey of allele-specific binding and expression over 1000-Genomes-Project individuals
Chen J, Rozowsky J, Galeev TR, Harmanci A, Kitchen R, Bedford J, Abyzov A, Kong Y, Regan L, Gerstein M. (2016) Nat Communi 7:11101
[2] AlleleSeq: analysis of allele-specific expression and binding in a network framework.
J Rozowsky, A Abyzov, J Wang, P Alves, D Raha, A Harmanci, J Leng, R Bjornson, Y Kong, N Kitabayashi, N Bhardwaj, M Rubin, M Snyder, M Gerstein (2011). Mol Syst Biol 7: 522 .

WEB: http://alleledb.gersteinlab.org/

CONTACT: [email protected]
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The package includes the AlleleDB pipeline developed in the Gerstein Lab at Yale.  This pipeline was built and applied to perform a uniform identification of SNVs associated with allele-specific binding and expression in 382 individuals from the 1000 Genomes Project [1]. It is based on the AlleleSeq pipeline (README follows below) developed earlier in the lab [2].  New developments include implementation of the beta-binomial test to estimate the statistical significance of the allele-specific event calls and a read filtering algorithm to account for the ambiguous mapping bias.  See the Methods section in [1] for details.  All newly added/modified scripts are prefixed with "alleledb_".

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Personal genome construction using vcf2diploid
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The pipeline requires Bowtie indices (for both haplotypes) of an individual's personal diploid genome and .fastq.gz file(s) of the functional assays (e.g. RNA-seq or ChIP-seq).  The personal diploid genome can be generated using the vcf2diploid tool [2] (http://alleleseq.gersteinlab.org/tools.html).  

The personal genomes of the 382 individuals from the study [1] are available at http://archive.gersteinlab.org/alleledb/personal_genomes/.  Below is an example command to build bowtie indices for each maternal and paternal haplotype of an individual (using the 1000 Genomes Project's individual HG00100 as an example):

bowtie-build --offrate 2 HG00100_pgenome_hg19/1_HG00100_maternal.fa,HG00100_pgenome_hg19/2_HG00100_maternal.fa,HG00100_pgenome_hg19/3_HG00100_maternal.fa,HG00100_pgenome_hg19/4_HG00100_maternal.fa,HG00100_pgenome_hg19/5_HG00100_maternal.fa,HG00100_pgenome_hg19/6_HG00100_maternal.fa,HG00100_pgenome_hg19/7_HG00100_maternal.fa,HG00100_pgenome_hg19/8_HG00100_maternal.fa,HG00100_pgenome_hg19/9_HG00100_maternal.fa,HG00100_pgenome_hg19/10_HG00100_maternal.fa,HG00100_pgenome_hg19/11_HG00100_maternal.fa,HG00100_pgenome_hg19/12_HG00100_maternal.fa,HG00100_pgenome_hg19/13_HG00100_maternal.fa,HG00100_pgenome_hg19/14_HG00100_maternal.fa,HG00100_pgenome_hg19/15_HG00100_maternal.fa,HG00100_pgenome_hg19/16_HG00100_maternal.fa,HG00100_pgenome_hg19/17_HG00100_maternal.fa,HG00100_pgenome_hg19/18_HG00100_maternal.fa,HG00100_pgenome_hg19/19_HG00100_maternal.fa,HG00100_pgenome_hg19/20_HG00100_maternal.fa,HG00100_pgenome_hg19/21_HG00100_maternal.fa,HG00100_pgenome_hg19/22_HG00100_maternal.fa,HG00100_pgenome_hg19/X_HG00100_maternal.fa HG00100_pgenome_hg19/HG00100_maternal > HG00100_pgenome_hg19/bowtie_build.maternal.log

bowtie-build --offrate 2 HG00100_pgenome_hg19/1_HG00100_paternal.fa,HG00100_pgenome_hg19/2_HG00100_paternal.fa,HG00100_pgenome_hg19/3_HG00100_paternal.fa,HG00100_pgenome_hg19/4_HG00100_paternal.fa,HG00100_pgenome_hg19/5_HG00100_paternal.fa,HG00100_pgenome_hg19/6_HG00100_paternal.fa,HG00100_pgenome_hg19/7_HG00100_paternal.fa,HG00100_pgenome_hg19/8_HG00100_paternal.fa,HG00100_pgenome_hg19/9_HG00100_paternal.fa,HG00100_pgenome_hg19/10_HG00100_paternal.fa,HG00100_pgenome_hg19/11_HG00100_paternal.fa,HG00100_pgenome_hg19/12_HG00100_paternal.fa,HG00100_pgenome_hg19/13_HG00100_paternal.fa,HG00100_pgenome_hg19/14_HG00100_paternal.fa,HG00100_pgenome_hg19/15_HG00100_paternal.fa,HG00100_pgenome_hg19/16_HG00100_paternal.fa,HG00100_pgenome_hg19/17_HG00100_paternal.fa,HG00100_pgenome_hg19/18_HG00100_paternal.fa,HG00100_pgenome_hg19/19_HG00100_paternal.fa,HG00100_pgenome_hg19/20_HG00100_paternal.fa,HG00100_pgenome_hg19/21_HG00100_paternal.fa,HG00100_pgenome_hg19/22_HG00100_paternal.fa,HG00100_pgenome_hg19/X_HG00100_paternal.fa HG00100_pgenome_hg19/HG00100_paternal > HG00100_pgenome_hg19/bowtie_build.paternal.log


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AlleleDB pipeline
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The main pipeline script is alleledb.sh, which is initialized with 11 positional command-line arguments, e.g:

./path-to-/alleledb.sh \
HG00100 \
HG00100_pgenome_hg19 \
/path-to-/HG00100_pgenome_hg19 \
/path-to-/HG00100_pgenome_hg19/HG00100.snp.calls.bed \
/path-to-/HG00100_rnaseq \
HG00100.1.M_111124_6 \
/path-to-/alleledb_pipeline \
/path-to-/alleledb_pipeline/PIPELINE.mk \
0.05 \
ase \
0

where the arguments:
arg1: individual/sample name; must conform with the suffix in the personal genome chromosome and indices names (e.g. 'HG00100' as in the example above)
arg2: personal genome (folder) name
arg3: path to the personal genome folder (no '/' at the end) containing bowtie indices, chromosome maps and hetSNP call files
arg4: path to the hetSNP calls .bed file, format e.g.:
        chr1    10582   10583   AG
        chr1    54675   54676   TC
        chr1    61986   61987   GA
arg5: path to the folder containing a functional dataset's read files (all .fastq.gz files contained in the folder will be pooled together and used for alignment and read counting)
arg6: assay-specific naming prefix
arg7: path to the folder alleledb_pipeline, containing the AlleleDB and AlleleSeq scripts
arg8: path to the AlleleSeq PIPELINE.mk file
arg9: FDR threshold
arg10: "ase" (allele-specific expression) or "asb" (allele-speciific binding). Depending on ASE or ASB, the arguments can slightly vary. Typically, because RNA-seq data has more coverage/reads, the FDR threshold can be lower (more stringent), e.g. 0.05 compared to a threshold for ChIP-seq of 0.1. Also note that the individual/sample name, should include the TFs for ASB, since you would want the folders to be on a per-individual per-TF basis.
arg11: the pipeline involves 8 steps/modules (each generating a subfolder under allelicbias-[arg3]-[arg1] and respective .log files); arg11 (normally with the value of "0": "run all steps") can be used for debugging/running a specific (1-8) step of the pipeline.
Running alleledb.sh with "-8" (as the only argument) will remove all files generated by module 8.


The personal genome folder should contain the following files with the filename format: (1) snp.calls (this tab-delimited file contains the snp calls) and (2) cnv_rd_[arg1]/rd.[arg1].txt (which is the read-depth file generated by the personal genome construction pipeline, AlleleSeq, http://alleleseq.gersteinlab.org/tools.html)
(1) snp.calls format e.g.:
1	10583	G	AA	AG	AG	PHASED
1	54676	C	TA	CA	TC	PHASED
1	61987	A	GT	AA	GA	PHASED
(2) rd.HG00100.txt:
chrm	snppos	rd
1	10583	33.2008
1	54676	0.760489
1	61987	1.17333

The pipeline requires bowtie and intersectBed to be in the path.
The main output of the pipeline is the file interestingHets.betabinom.txt, listing all HetSNVs associated with allele-specific behavior (that passed the FDR threshold) and formatted similarly to the interestingHets.txt file (see below) generated by the original AlleleSeq pipeline (with a new column - beta-binomial p-values).

The package contains example input files that can be used for a test run of the pipeline (one chromosome example ASE calculation for HG00100) under the ‘test’ directory.



===================================================================
The following is the README file for the AlleleSeq pipeline (v1.2a)
===================================================================

The Allele-specific SNP processing pipeline.  Written by Robert
Bjornson, Gerstein Lab, Yale University, in collaboration with Joel
Rozowsky.  Contact [email protected] or [email protected]
with questions or comments.

4/12/2012

There has been a major reworking to the innards of the Pipeline.
The motivation was to do as much of the processing as possible via
pipes, so that very little data is written to the disk.

In addition, two columns (Qval and KSval) were removed from the final output 
(interestingHets.txt), since they were no longer used.

The pipeline currently expects inputs as compressed fastq files, with 
the suffix .fastq.gz.  If you want it to work on other inputs, you'll
need to change both PIPELINE.mk and filter_input.sh

Each input file is transformed into a *.cnt file, which is a python pickle
of the snp counts for that file.  Those file are then combined and a report
is generated.

Because each mapping task runs two bowties, if you run the makefile in
parallel, run numprocs/2 tasks.
 
9/20/2010

The basic goal of the pipeline is to take a large collection of reads
representing ChIP-seq or RNA-seq data from a child in a trio with
known SNP locations, and detect snps that are significantly skewed to
one allele.  To do this, we first create references that represent the
childs' haplotypes (to the best of our ability).  We then map the reads
to both haplotypes, picking the best match for each read.  This is
done to eliminate the reference bias that would exist if we mapped to
the standard reference.  We then count the allele frequency observed
at each SNP position and look for imbalances, using false discovery
rate techniques to determine cutoffs.

-----------------------------------------------

The pipeline has six main inputs:
- one or more collections of unmapped reads.  In our case CHip-seq and
  RNA-seq from Illumina files.  The pipeline expects fastq format files,
  with the filename "*.fastq.gz".  All files matching this pattern will be
  combined into one dataset.  If you have something other than fastq.gz, e.g.
  bam or fastq, you will need add a preliminary conversion step.  

  As a sanity check, you can use the make target "check" to print out the set of input fastq files.

- a set of SNP locations for a trio.  The make variable SNPS should point to it.
  The pipeline expects this format:
chr	pos	ref	Mat	Pat	Child	Phase
1	114116078	A	TT	TT	TA	MUTANT
1	114117743	G	CG	GG	GG	HOMO
1	114120250	C	AC	AA	CA	PHASED
1	114120756	A	CA	CC	AC	PHASED

Mat and Pat are the maternal and paternal genotypes.
Child is child's genotype, ordered MatPat if phased.

Phase describes the phasing in the trio.  Possible values are:
HETERO (all three heterozygous, no phasing possible)
HOMO (child (subject) is homozygous, not an interesting snp position for our purposes)
PHASED (child heterozygous and at least one parent homozygous, so phasing was done)
MUTANT (child genotype is inconsistent with parents' genotype)

- a reference genome, used as a basis for creating the haplotype references for
the subject.

- a set of known genes, listing transcription start and stop, and exon
  coordinates, such as UCSC golden path.

- an optional set of ChIP-seq "binding sites", regions in the genome
where the transcription factor binds.  This will allow filtering of
SNP locations to just those within binding sites.  If BINDINGSITES is
set to a non-existent file in the makefile, no filtering will be done.

We used PeakSeek (J. Rozowsky) to determine binding regions.
The pipeline expects a file with no header in this format (only the first 3 fields will be read):
chr<tab>start<tab>end ...

The file need not be sorted by chr or start, but the format of chrom names must agree with 
the other files (usually chr#).

In the makefile, set BINDINGSITES to this file.  You can set it to an empty (but existent)
file, in which case depth will be set to 1.0 and no filtering will be done.

- a set of normalized read depth values for all snp locations from a
separate genomic sequencing experiment.  This reports the read depth
at that snp compared to overall coverage. This is used to filter out
locations with very low or high coverage, which would tend to indicate
copy number variation.  The file should be in this format:

chrm    snppos  rd
1       52066   0.902113
1       695745  0.909802
1       742429  0.976435

-----------------------------------------------

The processing follows these steps.

First, in a preliminary step (not part of the pipeline proper) the snp
calls and the reference genome are combined to generate two new
reference genomes, corresponding to the inferred maternal and paternal
haplotypes of the subject.  These two genomes are identical to the
original reference except in the SNP positions, which are changed to
be the alleles observed in the subject.  Alleles from SNPS that could
not be phased are randomly assigned maternal/paternal.  The script
CreateMirroredGenome.py was used to create these refs.

Pipeline proper: 

For each set of related reads: 
- The reads are trimmed, if necessary, to remove ends that contain
large numbers of errors and filtered to remove any reads containing
N's.

 - The filtered reads are mapped, using bowtie, to the maternal and
paternal reference genomes.  Bowtie is invoked with these flags:
--best --strata -v 2 -m 1, which returns only unique hits within a
minimum number of mismatches, up to two.  This was done to mimic the
semantics of eland.  By default, for simplicity, the included makefile
does this step inline.  Some degree of parallelism is possible via the
-j flag to make, assuming that you are running on a multicore system.
At Yale, we use a parallel harness developed locally called
SimpleQueue to do this step in parallel on multiple nodes.  See
PARALLEL.mk for the parallel pipeline, and
http://maguro.cs.yale.edu/mediawiki/index.php/SimpleQueue for an
explanation of the SimpleQueue tool.

 - The two sets of mapped reads are merged into a single set, with
each read represented at most once, using the better mapping from the
maternal or paternal reference.  If the two mappings for the same read
tie in quality, one is chosen at random.

 - Using SNP file and the mapped reads, allele counts are
generated for each SNP location.  The resulting counts file
contains the number of As,Cs,Gs, and Ts found in reads mapped over
each SNP-location.  Various other values are also generated for each
Het-SNP location, including reference allele, maternal/paternal allele
(if determinable), major and minor allele, and a binomial pvalue
assuming a 50/50 probability of sampling each of two alleles.

 - Explicit qvalues are calculated from the observed pvalues.

 - Using a qvalue cutoff, only those Het-SNPs showing significant 
bias are retained.  This is the filtered counts file for each dataset.

Once, for all counts files: 
- Using the list of genes and all filtered counts files, information
about all assymetric Het-SNPs from any of the datasets are grouped
together by gene.  The locations are annotated as being exonic or
intronic.  The information about each SNP includes: ref allele;
maternal and paternal genotype; phasing if determined; A,C,G,T counts;
biased-towards parent allele; Benjamini-Hochberg corrected pvalue,
explicitly calculated qvalue.

Programs used in our experiments:

Python 2.5.1
Bowtie 0.10.0.2 (Bowtie must be in your PATH)

Data files used:
SNP calls: Pilot 2 SNP calls from CEU.  
http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/knownGene.txt.gz Nov 2009.
Gene locations: UCSC GoldenPath.  
http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/knownGene.txt.gz  May 2009.
Human Reference: Standard HG18 without additional haplotypes

The actual pipeline driver is a makefile run by make in the directory
containing the read files: make -f <path to>/PIPELINE.mk.  That directory must also
contain directories called AltRefFather and AltRefMother, which
contain the haplotype references formatted for bowtie.

Make sure to edit PIPELINE.mk to set the variables as the top of the file,
especially BASE.

Main output Files:
counts.txt: Contains count information for all heterozygous SNP locations.
interestingHets.txt:  This file contains information about all SNP locations that passed
all these tests:
 - in a binding site
 - within range for cnv test
 - significantly assymetric by FDR test