Paper

https://www.biorxiv.org/content/10.1101/2023.03.21.533716v1

Installation

Before creating conda environment, please ensure packages are installed.

conda create -n LocusMasterTE python=3.6 future pyyaml cython=0.29.7 numpy=1.16.3 pandas=1.1.3 scipy=1.2.1 intervaltree=3.0.2

conda activate LocusMasterTE 
conda install -c bioconda htslib
pip install pysam==0.15.2
git clone https://github.com/jasonwong-lab/LocusMasterTE.git
cd LocusMasterTE
python3 setup.py build | python3 setup.py install 
LocusMasterTE bulk assign -h

Preprocessing Wrapper

LocusMasterTE provides a preprocessing wrapper script from Long-Read FASTQs to LocusMasterTE input. \ First, tools need to be installed. You may refer website for installation.

• Pychopper : https://github.com/epi2me-labs/pychopper
• Minimap : https://github.com/lh3/minimap2
• Samtools : https://www.htslib.org/
• FeatureCounts : https://rnnh.github.io/bioinfo-notebook/docs/featureCounts.html

\ 4 inputs are needed.

1. Genomic FASTA
2. Long Read FASTQ
3. Gene and TE GTF files
4. Path to Output Directory

Example code is below. \ bash LocusMasterTE/long_read_wrapper.sh hg38.fasta long_read.fastq.gz hg38.gtf output_path

If you have short-read FASTQ, here is the recommended code.

STAR --runThreadN 20 --genomeDir $outdir/STAR_index \
   --readFilesIn $short_fq1 $short_fq2 --readFilesCommand zcat --outFileNamePrefix STAR_output \
   --outSAMtype BAM Unsorted --outSAMstrandField intronMotif --outSAMattributes All --outSAMattrIHstart 0 \
   --outFilterMultimapNmax 100 --outFilterScoreMinOverLread 0.4 --outFilterMatchNminOverLread 0.4 --clip3pNbases 0 \
   --winAnchorMultimapNmax 100 --alignEndsType EndToEnd --alignEndsProtrude 100 DiscordantPair --chimSegmentMin 250 --twopassMode Basic
# sort by read name
samtools collate -o Aligned_sort_name.out.bam --output-fmt BAM Aligned_sort.out.bam

If you have short-read BAM file, BAM file needs to be sorted by read name. Run command below. And output bam can be readily input in LocusMasterTE. \ samtools collate -o Aligned_sort_name.out.bam --output-fmt BAM Aligned_sort.out.bam

Testing

bash LocusMasterTE/data/run_sample.sh

A BAM file (sample_alignment_sort.bam), annotation (annotation.gtf) and long read TPM file (long_read_data.txt) are included in LocusMasterTE/data folder. \ Recommended command line is written in bash file (run_sample.sh).

BAM file

When inputting BAM file, it should be sorted by READ NAME. Otherwise, LocusMasterTE does not work properly.
Aligned by coordinate also is not applicable.

Usage

• [LocusMasterTE bulk assign]

LocusMasterTE was built upon Telescope. Additional arguments are elaborated.

LocusMasterTE exclusive options:

  long_read
                        Mandatory argument. 
                        Path to long read file composed of three columns: "Geneid", "TPM", and "subF". 
                        "Geneid" represents TE individual names followed by TPM values in "TPM" coulmn. 
                        Belonging subfamily information from RepeatMasker database goes under "subF".
                        (default: None)
  Run Modes:

  --reassign_mode {exclude,choose,average,conf,unique,long_read}
                        Reassignment mode. After EM is complete, each fragment
                        is reassigned according to the expected value of its
                        membership weights. The reassignment method is the
                        method for resolving the "best" reassignment for
                        fragments that have multiple possible reassignments.
                        Available modes are: "exclude" - fragments with
                        multiple best assignments are excluded from the final
                        counts; "choose" - the best assignment is randomly
                        chosen from among the set of best assignments;
                        "average" - the fragment is divided evenly among the
                        best assignments; "conf" - only assignments that
                        exceed a certain threshold (see --conf_prob) are
                        accepted; "unique" - only uniquely aligned reads are
                        included. "long_read" - use long read to determine best hit.
                        NOTE: Results using all assignment modes are
                        included in the LocusMasterTE report by default. This
                        argument determines what mode will be used for the
                        "final counts" column. (default: exclude)

Model Parameters:

  --rescue_short RESCUE_SHORT
                        To rescue features only captured by short, 
                        values can be given to 0 expression captured in long read.
                        (default: 0)
  --long_read_weight {float}
                        Weights on long-read information; No limited numeric ranges.
                        Higher number is recommended when matched tissue or cell type long-read is inputted.
                        Lower number is recommended when using different tissue samples.
                        (default: 1)
  --prior_change {all,theta,none}
                        Integration of TPM counts from long reads. 
                        All represents change in both pi and theta.
                        Change in theta influences only multimapping counts.
                        None is equivalent to not integrating long read
                        (default: all)

Arguments from Telescope

Output

LocusMasterTE has three main output files: the transcript counts estimated via EM (LocusMasterTE-TE_counts.tsv).\ The count file is most important for downstream analysis.