MiSeq- Getting the sequence of bases from the Alignment Folder

[Krina796]

Hello MiSeq community,

I'm using the MiSeq, and trying to figure out how to see the actual bases in the sequence that's been written to the Alignment folder. How do I do this?

If I'm understanding correctly, the bases are written to BAM files in binary format. Then, these BAM files must be converted into the corresponding alphabetical bases using a third party software, such as IGV or BAM viewer, which is not provided by Illumina. At our lab, it's very difficult to get permission to download outside software on our computers, even free software like IGV. Is there any other way to see the actual sequence using just Illumina-provided software?

Any and all help is appreciated, I'm very new to this, and am trying to figure out if it's absolutely necessary to download the third party software.


Thank you,
Krina

[GenoMax]

Krina: You are probably using the alignment workflow on the MiSeq which results in the generation of BAM files. You can potentially rerun the analysis choosing "FASTQ only" workflow on MiSeq reporter that will generate sequence files only (no alignments). http://supportres.illumina.com/docum...15042322_a.pdf

But before you do that have you looked in the "D:\Illumina\MiSeqAnalysis\Flowcell_ID\Data\Intensities\BaseCalls" folder to see if the sequence files are there?

There may be a better/simpler answer (we do not use the onboard analysis on our MiSeq's so I am not 100% sure) but while you wait to hear from someone give the above a try.

[Krina796]

Hi GenoMax,

Thank you very much for your help! I just read through the document you sent to me, and I'm not sure if I'm understanding entirely.

Using the MiSeq Reporter's "FASTQ only" workflow, can you actually see the long list of bases of the genome you're sequencing? And is the "alignment" process when you compare the sequence in the FASTQ file, which is converted into a BAM file and then another readable file, to the reference genome? (i.e. the "alignment" process is what requires the third party software, but you don't need third party software to simply see the sequence of your sample?)

Thanks again for your help! I apologize if my questions are confusing.

Best,
Krina796

[GenoMax]

Using the MiSeq Reporter's "FASTQ only" workflow, can you actually see the long list of bases of the genome you're sequencing?

"FASTQ only" work flow will generate sequences from the fragments but will not do any alignments. You would basically get a file with sequences (millions of them) from each fragment/cluster in your sample in FASTQ format. There will be two separate files (with *R1* and *R2* in the name) if you do a paired-end sequencing run (*R1* only for a single-end run).

And is the "alignment" process when you compare the sequence in the FASTQ file, which is converted into a BAM file and then another readable file, to the reference genome? (i.e. the "alignment" process is what requires the third party software, but you don't need third party software to simply see the sequence of your sample?)

If you provide MiSeq reporter with a reference sequence (or use one of the standard ones provided (e.g. human, mouse) ) then the MiSeq reporter does an alignment of all reads that pass QC and generates the BAM file. The BAM file is in binary format and is not human readable. You do not need any third party software if you are happy with the alignments that MiSeq produces.

[GenoMax]

Are you the one running the MiSeq or are you using a core facility where someone else is responsible for the run?

[Krina796]

Thank you for your help, GenoMax!

Are you the one running the MiSeq or are you using a core facility where someone else is responsible for the run?

I'm currently the one trying to figure out how to run the MiSeq, this isn't yet a core facility.