How do I run UNAGI with multiple samples?

[chaturvedi-lab]

Hi there,

I have nanopore cDNA data from four samples and I would like to run UNAGI on these samples. How do I do this? Do I: 1) run UNAGI individually for each sample? If so how do I concatenate the results? 2) Combine the reads from 4 samples and run UNAGI?

Thanks, Sam

[Kyomari]

To run UNAGI on your nanopore cDNA data from four samples, you can follow these general steps:

Preprocessing: Adapter trimming and quality control: Use a tool like Porechop or Guppy to remove adapters and trim low-quality reads. You can also use tools like NanoPlot or FastQC to assess the quality of the reads before and after trimming.

Alignment: Use a tool like minimap2 to align the trimmed reads to the reference genome or transcriptome. This will generate a BAM file for each sample.

Transcript quantification: Run UNAGI on each sample separately using the aligned BAM file as input. UNAGI will quantify the expression of transcripts based on the reads that align to them.

I hope this helps.

[chaturvedi-lab]

Thank you for the quick reply! I just want to confirm one last thing. Do I run UNAGI with the bam files as follows (similar to the fastq file):

unagi -i sample.sorted.bam -g genome.fasta -o outdir

[Kyomari]

Yes. Here, sample.sorted.bam is the aligned BAM file for your sample, genome.fasta is the reference genome or transcriptome in FASTA format, and outdir is the directory where UNAGI will output the results.

[chaturvedi-lab]

Hi there,

I ran unagi with the sorted bams and I get the following error:

[2023/05/05 - 15:09:25] Reading the input file [2023/05/05 - 15:09:25] The input file must be in the fastq or fastq.gz format.

Thanks, Sam

[Kyomari]

Based on the error message, it seems like unagi is expecting a fastq or fastq.gz file as input, but instead it's being given a sorted BAM file. You'll need to convert your sorted BAM file to a fastq file. You can do this using a tool like samtools, which can convert a BAM file to a fastq file. Here's an example command in lua:

samtools fastq input.bam > output.fastq

Replace input.bam with the path to your sorted BAM file and output.fastq with the desired name and path for the resulting fastq file.