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Telescope

Before starting today, you may need to:

Sync your copy of the teabiscuit repository using git

cd ~/teabiscuit
git pull

Activate your teabiscuit environment that we made on Monday:

conda activate teabiscuit

Alignment

SAM/BAM files

The SAM/BAM format is a standardized file format for representing sequence alignments to a reference genome. It is able to include all the data needed to describe an alignment and the original input FASTQ files can be reconstructed (lossless). SAM files are tab-delimited text files and can be viewed with common command-line tools. BAM files are compressed (zipped) binary files that may be sorted and indexed for fast retrieval of alignments overlapping a genomic region.

The samtools package is useful for working with SAM/BAM format files.

SAM/BAM files can also be used to store unaligned data (uBAMs)

CRAM files are able to achieve better compression than BAM files by storing reads aligned to a reference sequence and only storing the bases that differ from the reference. One may also opt for controlled loss of data for even better compression, i.e. eliminating read names or qualities.

Viewing BAM files

Activate teabiscuit environment:

samtools view ENCSR693KOP/Aligned.out.bam | less -S

This python code will print out alignments for all the reads with 500 alignments:

import pysam
af = pysam.AlignmentFile('ENCSR693KOP/Aligned.out.bam')
iter = af.fetch(until_eof=True)
for aseg in iter:
    if aseg.has_tag('NH') and aseg.get_tag('NH')==500:
        print(aseg.to_string())

Visualizing alignments

Tools for visualizing alignments:

Download and install IGV

Go to https://software.broadinstitute.org/software/igv/download

Follow the directions for your OS. I recommend the "Java included" versions unless you have a good reason not to.

Choose Genome

We are going to be viewing annotations and alignments for hg38

Load data tracks

tl;dr Download this IGV session file locally igv_session.STAR.xml and open it using "File > Open Session..."

Data tracks are loaded using the "File" menu. Data can be loaded from a local file (on your computer) using "File > Load from File..."; from a URL ("Load from URL...") or from a public server ("Load from server...").

We are going to load from a URL so that you don't have to download all the files locally. The tracks we will load are:

GENCODE annotation v38

URL: http://nixonlab-cluster.hopto.org/efs/projects/teabiscuit.git/02-telescope/gencode.v38/gencode.v38.ALL.annotation.gtf.gz Index: http://nixonlab-cluster.hopto.org/efs/projects/teabiscuit.git/02-telescope/gencode.v38/gencode.v38.ALL.annotation.gtf.gz.tbi

Telescope annotation

URL: http://nixonlab-cluster.hopto.org/efs/projects/teabiscuit.git/02-telescope/retro.hg38.v1.gtf

Alignment (BAM file)

URL: http://nixonlab-cluster.hopto.org/efs/projects/teabiscuit.git/02-telescope/ENCSR693KOP/Aligned.out.sortedByCoord.bam.bai Index: http://nixonlab-cluster.hopto.org/efs/projects/teabiscuit.git/02-telescope/ENCSR693KOP/Aligned.out.sortedByCoord.bam

RepeatMasker tracks

Go to "File > Load From server" Navigate the nested structure with the drop-downs