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Learn how Kallisto works and how to use it.
Introduction
Kallisto is a super fast tool for transcript quantification. It gets it speed by skipping the alignment step. Instead of actually aligning reads to a transcriptome, it identifies transcripts that a read is compatible with in order to quantify the transcript. This process is called pseudoalignment/pseudomapping.
Typically, after quality assessment, the first two steps of RNA-Seq workflow are mapping, followed by quantification of genes/transcripts. Mapping with a mapper can take a 6+ hours for a very large file. Quantification can then take several more hours. Kallisto avoids the mapping step and through a process called pseudoalignment/ pseudomapping, it proceeds directly to the quantification step. It is reported that Kallisto can quantify 30 million human reads in less than 3 minutes on a mac laptop.
Get your data
Six raw data files have been provided for all our further RNA-seq analysis:
- c1_r1, c1_r2, c1_r3 from the first biological condition
- c2_r1, c2_r2, and c2_r3 from the second biological condition
Get set up for the exercises
| Code Block |
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| title | Get set up for the exercises |
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cds
cd my_rnaseq_course
cd day_2/kallisto_exercise |
Kallisto is available on lonestar6- check for it.
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| title | Look for kallisto module |
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module load biocontainers
module load kallisto |
| Code Block |
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| title | Find the singularity command |
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type kallisto |
Part 1. Create a index of your reference
NO NEED TO RUN THIS NOW- YOUR INDEX HAS ALREADY BEEN BUILT!
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singularity exec ${BIOCONTAINER_DIR}/biocontainers/kallisto/kallisto-0.45.0--hdcc98e5_0.simg kallisto index -i transcripts.idx transcripts.fasta |
Part 2. Quantify transcripts using kallisto
| Warning |
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| title | Submit to the TACC queue or run in an idev shell |
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Create a commands file and use launcher_creator.py followed by sbatch. | Code Block |
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| title | Put this in your commands file |
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| nano commands.quant
singularity exec ${BIOCONTAINER_DIR}/biocontainers/kallisto/kallisto-0.45.0--hdcc98e5_0.simg kallisto quant -i ../reference/transcripts.idx -b 100 -o GSM794483_kallisto ../data/GSM794483_C1_R1_1.fq ../data/GSM794483_C1_R1_2.fq
singularity exec ${BIOCONTAINER_DIR}/biocontainers/kallisto/kallisto-0.45.0--hdcc98e5_0.simg kallisto quant -i ../reference/transcripts.idx -b 100 -o GSM794484_kallisto ../data/GSM794484_C1_R2_1.fq ../data/GSM794484_C1_R2_2.fq
singularity exec ${BIOCONTAINER_DIR}/biocontainers/kallisto/kallisto-0.45.0--hdcc98e5_0.simg kallisto quant -i ../reference/transcripts.idx -b 100 -o GSM794485_kallisto ../data/GSM794485_C1_R3_1.fq ../data/GSM794485_C1_R3_2.fq
singularity exec ${BIOCONTAINER_DIR}/biocontainers/kallisto/kallisto-0.45.0--hdcc98e5_0.simg kallisto quant -i ../reference/transcripts.idx -b 100 -o GSM794486_kallisto ../data/GSM794486_C2_R1_1.fq ../data/GSM794486_C2_R1_2.fq
singularity exec ${BIOCONTAINER_DIR}/biocontainers/kallisto/kallisto-0.45.0--hdcc98e5_0.simg kallisto quant -i ../reference/transcripts.idx -b 100 -o GSM794487_kallisto ../data/GSM794487_C2_R2_1.fq ../data/GSM794487_C2_R2_2.fq
singularity exec ${BIOCONTAINER_DIR}/biocontainers/kallisto/kallisto-0.45.0--hdcc98e5_0.simg kallisto quant -i ../reference/transcripts.idx -b 100 -o GSM794488_kallisto ../data/GSM794488_C2_R3_1.fq ../data/GSM794488_C2_R3_2.fq |
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| title | Use this Launcher_creator command |
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| launcher_creator.py -n kallisto -t 01:00:00 -j commands.quant -q normal -a OTH21164 -l kallisto_launcher.slurm -m "module load biocontainers;module load kallisto" |
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Output files:
abundances.tsv: A tsv file containing raw read count and a normalized expression value for each transcript.
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