Real Use Cases

Estimating allele-specific expression from human RNA-seq data

1. Process reference data

We need to first extract transcript information from the reference genome and gene annotation. Most importantly, EMASE requires the list of transcript ID’s and which gene each transcript belong to.:

prepare-emase -G ${REF_FASTA} -g ${REF_GTF} -o ${REF_DIR} -m --no-bowtie-index

‘prepare-emase’ generates the following files for the reference genome:


2. Build an individualized genome

We assume there is a vcf file that contains phased variant information for every sample of your population. Unless we know which allele is M(aternal) or P(aternal), we are going to distinguish two alleles with suffices, L(eft) and R(ight). We also recommend to use different ${SAMPLE_DIR} for each sample:

python -r ${REFERENCE_FASTA} \
                                       -i ${INDEL_VCF} \
                                       -s ${SNP_VCF} \
                                       -d ${SAMPLE_DIR} \
                                       -o L.fa \
python -r ${REFERENCE_FASTA} \
                                       -i ${INDEL_VCF} \
                                       -s ${SNP_VCF} \
                                       -d ${SAMPLE_DIR} \
                                       -o R.fa \

3. Individualize gene annotation

We want to incorporate individual variation into the gene annotation too so we can build individualized transcriptome in the following step:

python -s 4 -e 5 -c 1 -t 9 \
                             -d ${SAMPLE_DIR} \
                             -o L.gtf \
                             -C ${SAMPLE_HAP1_ID_IN_VCF}_comments.txt
                             ${REFERENCE_GTF} \
python -s 4 -e 5 -c 1 -t 9 \
                             -d ${SAMPLE_DIR} \
                             -o R.gtf \
                             -C ${SAMPLE_HAP1_ID_IN_VCF}_comments.txt
                             ${REFERENCE_GTF} \

4. Create a personalized diploid transcriptome

From L.fa, R.fa, and the corresponding gtf files, we are going to create diploid transcriptome and other information that EMASE requires. We assume bowtie v1.0.0 or newer is available.:

prepare-emase -G ${SAMPLE_DIR}/L.fa,${SAMPLE_DIR}/R.fa -s L,R -o ${SAMPLE_DIR}

This will generate the following files:


5. Align RNA-seq reads against the diploid transcriptome

Although EMASE is a flexible framework for many other alignment strategies, the current version of EMASE was most intensely tested with bowtie1 transcriptome alignments with the following parameters:

bowtie -q -a --best --strata --sam -v 3 ${SAMPLE_DIR}/bowtie.transcriptome ${FASTQ_FILE} \
    | samtools view -bS - > ${SAMPLE_DIR}/bowtie.transcriptome.bam

6. Convert bam file into the emase format

EMASE runs on an alignment profile of three-dimensional incidence matrix. We convert an alignment file (bam) to the EMASE format using the following script:

bam-to-emase -a ${SAMPLE_DIR}/bowtie.transcriptome.bam \
             -i ${REF_DIR}/ \
             -s L,R \
             -o ${SAMPLE_DIR}/bowtie.transcriptome.h5

7. Run EMASE

Now we are ready to run EMASE:

run-emase -i ${SAMPLE_DIR}/bowtie.transcriptome.h5 \
          -g ${REF_DIR}/emase.gene2transcripts.tsv \
          -L ${SAMPLE_DIR}/ \
          -M ${MODEL} \
          -o ${SAMPLE_DIR}/emase \
          -r ${READ_LENGTH} \

‘run-emase’ outputs the following files as a result:


Estimating allele-specific binding from ChIP-seq data

We assume you have a set of individualized genome and annotation files, in this example, S1 and S2, created by Seqnature package. We also assume you have a bed file that specifies genomic regions of your interest. First, you need to convert your bed file into a simple gtf format:

bed-to-gtf -i targets.bed -o targets.gtf

The targets.gtf files should be modified according to the strains of our interest:

python -s 4 -e 5 -c 1 -t 9 -o S1.gtf -C S1_comments.txt targets.gtf S1
python -s 4 -e 5 -c 1 -t 9 -o S2.gtf -C S2_comments.txt targets.gtf S2

Finally, run:

prepare-emase -G S1.fa,S2.fa -g S1.gtf,S2.gtf -s S1,S2 -o S1xS2

This will store the following files in the folder ‘S1xS2’:


Now you can align your RNA-seq reads against the pooled bowtie index of target region:

bowtie -q -a --best --strata --sam -v 3 S1xS2/bowtie.transcriptome ${FASTQ_FILE} \
    | samtools view -bS - > S1xS2/bowtie.transcriptome.bam

Next, we convert the alignment file into a format that EMASE use for running EM algorithm:

bam-to-emase -a S1xS2/bowtie.transcriptome.bam \
             -i S1xS2/ \
             -s S1,S2 \
             -o S1xS2/bowtie.transcriptome.h5

It is now ready to run emase. We assume the read length is 100bp:

run-emase -i bowtie.transcriptome.h5 -L S1xS2/ -M 4 -c

Deconvolving human and mouse gene expression from Patient-Derived Xenograft (PDX) models

Coming soon!

Estimating allele-specific expression from a F1 sample

Coming soon!