Problems with kit 600 cycles

[Mario Moreno]

Hi,
We were using kit 600 cycles v3 for library made in truseq RNA in Miseq, but the quality of the results was bad in the ends and the length of the reads was 240 pb in average. We saw in illumina that the quality was good in comparison with 500 cycles.
Maybe the problem is that this kit isn’t compatible with truseq library or this kit is exclusive with nextera library, we don’t know this.
Help me please.
Thanks!

[GenoMax]

The problem here may not be the kit but your library. It is possible that inserts in your library are shorter than you expect them to be.

I assume "average length of reads 240 bp" is after trimming?

[Mario Moreno]

Hi,
Yes, the "average length of reads 240 bp" is after trimming.
you know if the kits is compatible with truseq library or this kit is exclusive with nextera library?

Thanks

[microgirl123]

The 600-cycle kit is compatible with TruSeq - we've had great results with it on TruSeq libraries. What does your Bioanalyzer trace look like?

[pmiguel]

Which TruSeq library kit?
With minor modifications, a 600 cycle kit would work for most TruSeq libraries. For some values of "minor"...

--
Phillip

[Mario Moreno]

Thanks you for your prompt replies.
I am wondering if you use the same protocol or perform variations in the step of fragmentation of the mRNA ( Elution 2-Frag-Prime: 94°C for 5 min.) or in other step.
Thanks

[pmiguel]

Thanks you for your prompt replies.
I am wondering if you use the same protocol or perform variations in the step of fragmentation of the mRNA ( Elution 2-Frag-Prime: 94°C for 5 min.) or in other step.
Thanks

For RNA we use 1-2 minutes instead of 8 minutes for fragmentation. Also we use lower ratios of sample:ampure for clean-ups. But to figure the ratios out we calibrate each Ampure bottle. (We don't a gel cut purification of samples, if you do a gel cut, you might not need to modify the Ampure amounts.)

But the trick is, you have to do both -- fragment shorter time and use less ampure.

You can tell how you did by running the final, amplified library on a bioanalyzer chip.

Look at the left-hand side of the peak.

The very left-most size will seed the majority of your clusters. So if you see anything at all at 370bp or less, then you will get lots of reads that contain adapter and/or read all the way through into the flow cell oligo attachment. For best results you want the left side of your peak to be >720bases. That way your forward and reverse reads will both be providing novel sequence. But to get to 600 base inserts you may need to fragment less than 1 minute.

Obviously, if you remove the shorter insert amplicons, then you bias the library away from short (>500 nucleotide) transcripts. So there is a cost...

--
Phillip

[DJ Flowcell]

Hi Mario,

Yeah you would definitely run into problems with such short libraries. As Phillip points out, if you are running 2x300bp runs, you will need your average total library size to be at least 420bp - 300bp for the actual library and 120bp for the adapters (60bp each). Otherwise, you will run into adapter sequences.

[Mario Moreno]

Thanks you for your prompt replies.
It has helped me a great help.
Soon will perform the modifications to make the libraries with larger means.
When you have the results back to comment
Thanks!

[snetmcom]

For RNA we use 1-2 minutes instead of 8 minutes for fragmentation. Also we use lower ratios of sample:ampure for clean-ups. But to figure the ratios out we calibrate each Ampure bottle. (We don't a gel cut purification of samples, if you do a gel cut, you might not need to modify the Ampure amounts.)

But the trick is, you have to do both -- fragment shorter time and use less ampure.

You can tell how you did by running the final, amplified library on a bioanalyzer chip.

Look at the left-hand side of the peak.

The very left-most size will seed the majority of your clusters. So if you see anything at all at 370bp or less, then you will get lots of reads that contain adapter and/or read all the way through into the flow cell oligo attachment. For best results you want the left side of your peak to be >720bases. That way your forward and reverse reads will both be providing novel sequence. But to get to 600 base inserts you may need to fragment less than 1 minute.

Obviously, if you remove the shorter insert amplicons, then you bias the library away from short (>500 nucleotide) transcripts. So there is a cost...

--
Phillip

very helpful. I also recommend shorter fragmentation times.
I am curious what Ampure ratios do you typically test.

[nucacidhunter]

In my experience size cut will be around 160 bp and 100 bp for 1x and 1.2x, respectively. It is easy to test the size cut without a library if you have a Covaris. Shear some DNA and size select (left size selection) 100 ng of shear with various ratios of beads and after elution, run size selected sample and input shear on TapeStation or Bioanlyzer.
I have not seen differences with different batches of AMPure beads in practice to justify calibration. I have only read about it in 454 DNA library prep kit manual which recommends titration with DNA ladder. Sheared DNA is much better than ladder for calibration to see the exact size cut because fragment size distribution is continuous like the libraries. If you are worried about titration, the alternative is SPRIselect which has already been factory calibrated and it only cost little more.

[JBKri]

Hi Mario,

Yeah you would definitely run into problems with such short libraries. As Phillip points out, if you are running 2x300bp runs, you will need your average total library size to be at least 420bp - 300bp for the actual library and 120bp for the adapters (60bp each). Otherwise, you will run into adapter sequences.

If we do run a library with a short insert (~260 bp) with a 2x300 cycles kit, would it cause any problems other than reading into the adapters? Would it affect the quality of the index read, or read2?

Jon

[nucacidhunter]

If we do run a library with a short insert (~260 bp) with a 2x300 cycles kit, would it cause any problems other than reading into the adapters? Would it affect the quality of the index read, or read2?

Jon

No, it won't affect any of those.

[nbogard]

What if the number of cycles is longer than the insert + adapter + flow cell oligo?

[nucacidhunter]

The overall quality displayed in SAV will be very low. FastQC plots also will show that quality is dropping as the cycle number increases. The quality of insert or index reads will not be affected and should be high after trimming adapters.