Miseq questions from a newbie

[Neiltje]

Hi,

I'm new in the sequencing world (apart from standard courses in university).
I've prepared an RNAseq library using the Scriptseq protocol from Epicentre. Together with a co-worker we sequenced it on a V2 150bp (300 cycles) cassette. Now I'm learning more about sequencing, I'm wondering why we used this cassette and not another one.

Next week, I'm going to prepare a library of small RNA's for sequencing. The fragment size will be around 147 bp (sequence itself + adaptor + indices). But my boss told me to order the v2 cartridge of 50 cycles. Now I'm wondering how long the reads will be that come out of it. If it is 2 x25bp, won't the Miseq read only the adaptor and never the sequences of interest itself?

I'm sorry if these are really silly question, I'm just getting the hang of it.

[Neiltje]

Oh and another question.
Why is sequencing of a PCR fragment on a Miseq overkill?

[GenoMax]

Number of cycles is equal to number of bases you are going to get for each sequence read. So your 50 cycle single-end run will yield 50bp from (let us just say from left end) of a fragment. If you did a paired-end 50 cycle run then that will give you an additional 50 bp of sequence (from the right end) of the SAME fragment.

So for a ~150 bp fragment

      DNA Fragment         ----------------------------
      Single-end (50 bp)   --------->
                                             <--------- Paired-end (50 bp)

Sequencing PCR fragments is something people do all the time on MiSeq. It is a popular application for MiSeq.

[Neiltje]

So my thinking was right, 50 cycles for a fragment of +/- 147 bp, will not cover the sequence in the middle? 50 bp from the right and 50 bp from the left, still leaves 47 bp in the middle.


About the PCR fragment: if you do primerdesign for the fragment you're interested in, you need to design primers compatible to your DNA + adaptor and index sequence(s). Aren't this really long primers?

[GenoMax]

If you want the fragments to overlap in the middle then you will need to do a longer (100 bp) paired-end run (which may require a different kit). There is software available that can then stitch the reads together to form a single long read (MiSeq software can do this on the machine or you can use other programs like FLASH).

Here are example threads for primer design:

http://seqanswers.com/forums/showthread.php?t=20373

http://seqanswers.com/forums/showthread.php?t=33556

http://seqanswers.com/forums/showthread.php?t=17816

[microgirl123]

"About the PCR fragment: if you do primerdesign for the fragment you're interested in, you need to design primers compatible to your DNA + adaptor and index sequence(s). Aren't this really long primers?"

Yes but there are ways around it. You wouldn't want to sequence a single PCR amplicon on the MiSeq - it's appropriate for mixed amplicons such as mixed 16s sequences.

[microgirl123]

"The fragment size will be around 147 bp (sequence itself + adaptor + indices). But my boss told me to order the v2 cartridge of 50 cycles. Now I'm wondering how long the reads will be that come out of it. If it is 2 x25bp, won't the Miseq read only the adaptor and never the sequences of interest itself?"


This is a really small insert - your adapter/index sequences are ~130 bp or more. The adapters are a combination of hybridization sites, where the library binds to the flow cell, index sequences, and priming sites for both reads and for index reads. The priming sites for the reads are immediately adjacent to your insert so sequencing starts right at the beginning of your insert not at the beginning of your adapter.

[Neiltje]

"The fragment size will be around 147 bp (sequence itself + adaptor + indices). But my boss told me to order the v2 cartridge of 50 cycles. Now I'm wondering how long the reads will be that come out of it. If it is 2 x25bp, won't the Miseq read only the adaptor and never the sequences of interest itself?"


This is a really small insert - your adapter/index sequences are ~130 bp or more. The adapters are a combination of hybridization sites, where the library binds to the flow cell, index sequences, and priming sites for both reads and for index reads. The priming sites for the reads are immediately adjacent to your insert so sequencing starts right at the beginning of your insert not at the beginning of your adapter.

Ok, thank you
I just discovered Illumina has a lot of online training video's. They also clarify a lot.