Demystifying the MiSeq!

[microgirl123]

I work in a core facility with that's just gotten into next-gen sequencing with the purchase of a MiSeq. It's been a learning experience! I'm trying to write a short, but detailed, introduction to how MiSeq sequencing actually works and what a constructed library looks like, including detailed explanations of what's going on with the TruSeq adapters.

I'm stuck at a couple of key points though and I hope someone will have some suggestions! The first concerns when and how the index sequence is read on the MiSeq. My understanding is that, after cluster generation, the clusters are all attached to the flow cell by the universal P5 (not indexed) adapter. The sequencing primer that is hybridized on is attached at the priming site on the indexed adapter (P7). Sequence is then generated downward toward the flow cell until Read 1 is complete.

Somewhere in between Read 1 and Read 2 the index read occurs and the Illumina website doesn't seem to have details on this. I assume that turnaround and cluster regeneration must happen first so that the index is now at the "top" of the cluster rather than just above the flowcell binding site. Is there a separate primer for the index read that is located within the P7 attachment region? That would mean that this primer would anneal, the index reads would occur, all that would be washed off, and the Read 2 primer hybridized? If anyone has some input on this or great websites that explain things, please post them!!!!!

Also, all of these questions came about due to difficulties people are having in removing adapter dimer sequences from their data. The current thought is that in Read 1 (originating from the priming site on the Universal Adapter), data should never begin with the reverse complement of the Indexed Adapter. In Read 2 (originating from the priming site on the Indexed Adapter), data should never begin with the reverse complement of the Universal Adapter.

Sorry this is so long, and thank you to anyone with any thoughts and ideas!

[mastal]

Demystifying the MiSeq!

Hi microgirl123,

There are various websites as well as Illumina documents with helpful diagrams showing where the TruSeq index sequences go.

Some of the ones I have found most helpful are:

The 'Illumina - all flavors' web page from the U. Texas at Austin Genome Sequencing Facility:

https://wikis.utexas.edu/display/GSAF/Illumina+-+all+flavors

The Tufts University Core Facility have a document called "Illumina TruSeq DNA Adapters De-Mystified" by James Schiemer, at

http://genomics.med.tufts.edu/documents/protocols/TUCF_Understanding_Illumina_TruSeq_Adapters.pdf

Best wishes,
Maria

[microgirl123]

Thanks, Maria. I hadn't seen the U. Texas info. The Tufts adapter info has been really helpful.

Also, does anyone know what the FASTQ files really are? Are they the sequence of bases that are fluorescing or are they the reverse complement of those bases, i.e. the bases actually present on the strand being sequenced?

[krobison]

Also, does anyone know what the FASTQ files really are? Are they the sequence of bases that are fluorescing or are they the reverse complement of those bases, i.e. the bases actually present on the strand being sequenced?

Quality scores increase from left to right; therefore the calls are what fluorescent nucleotide was added.

[microgirl123]

Quality scores increase from left to right; therefore the calls are what fluorescent nucleotide was added.

I'm sure I'm being dense here, but I have no idea what this means. What I'm trying to ask is, when the MiSeq makes its basecalls and creates FASTQ files, is it telling me what the fluorescent molecule was (let's say a T) or is it telling me what the fluorescent molecule bound to (an A). Of course, there's two steps here, the basecalling and the FASTQ file creation, so either of those steps could process the fluorescence to report what is actually on the strand (the A).

I'm really having trouble deciding which strand of DNA is being sequenced when and what the software is actually reporting!

[JackieBadger]

Illumina support are really good and will have no problem explaining this all to you over the phone

[mastal]

Demystifying the MiSeq!

I think the nucleotides reported in the fastq files are the nucleotides being incorporated.

But just at the moment I can't remember if I have any reference that specifically states this.

Maria

[kmcarr]

MG,

Have a look at this document from Illumina which pretty clearly describes the structure of the library molecules and relative position/orientation of the sequencing primers.

After cluster generation, linearization and blocking the Read 1 sequencing primer (SP) is annealed. After completion of read 1 sequencing the nascent strand is denatured and the Index SP is annealed; it anneals to the same strand as the read 1 SP. After the index read is complete, and IF you are performing a paired end protocol, the resynthesis (a.k.a. cluster regeneration or turn around) chemistry is performed. After resynthesis the Read 2 SP is annealed. The Read 2 SP is actually the reverse complement if the Index SP.

[microgirl123]

Thanks, kmcarr and Maria. I never thought to look for Illumina documents on anything but the MiSeq! Also, my second query to Illumina tech support yielded better results. The FASTQ files are reporting the sequence of the fluorescent, incorporated nucleotides, which are really the sequence of the original strand because what is being sequenced is the reverse complement! This all makes my brain hurt!