Chapter 6 Week 2 Part B - Quantification with Kallisto

Now that our adapters and poor quality sequences have been removed from our reads we can proceed to quantification.

6.0.1 Transcript quantification with Kallisto

Kallisto is a tool that can quantify transcript expression using the pseudoalignment method. Given it doesn’t rely on alignments, we can process reads with kallisto directly. In the final part of the workshop we will generate transcript quantifications for our sample.

Kallisto has several functions. The function we will use is the quant function (kallisto quant OPTIONS R1 R2). We need to provide the tool the following flags.

• -i: This must point to ~/week_2/resources/indexes/kallisto/WBcel

• -o: Point this to the output directory

Exercise 5

• Use kallisto quant to generate transcript quantifications.

  • Read the Kallisto manual to ensure you do this correctly. You can use a loop or write a command to manually achieve this. (https://pachterlab.github.io/kallisto/manual)

    • This will generate a .tsv file in your output directory

• Use cat and sort to identify the transcripts with the greatest TPM. Hint: This is in the fifth column of the file

kallisto quant -i /apps/data/bms5021/week_2/indexes/kallisto/WBcel -o ~/week_2/trimmed_reads_TrimGalore sample_1_R1.fq sample_1_R2.fq sample_2_R1.fq sample_2_R2.fq sample_3_R1.fq sample_3_R2.fq sample_4_R1.fq sample_4_R2.fq sample_5_R1.fq sample_5_R2.fq sample_6_R1.fq sample_6_R2.fq