modification of ScriptSeq-RNA v2 protocol to accommodate v3 chemistry

[exo]

Dear all,

Just wanted to share with you some possible modifications i will be implementing in the ScriptSeq RNA v2 protocol for sequencing using the V3 chemistry.

1- I was thinking to reduce the RNA fragmentation time to 3 or 4 min at 85 deg (down from 5min at 85 deg).

2- Increase cDNA synthesis time using random hexamers to 60min at 42 deg using AMV (up from 20min at 42 deg)

3- cDNA purification using 0.9x beads (instead off 1.8x previously) - too rough at this step?

4- RNA seq lib purification using 0.85x beads (instead off 1x previously)

What do you think about the following modifications?

I have been having problems lately using the protocol as it is with the v3 chemistry (2x300bp read length).
My library had an average fragment length of 400bp ending resulting in a mean read length of 180bp (which is quite short!).

I just wanted to make sure those steps make sense.

Best,
E

[Olaf Blue]

Hi Exo-

The modifications look ok and are not that much of a deviation from the protocol. They should be fine.

A couple of other things - the StarScript RT used in ScriptSeq library prep is an AMV....second, the PCR amplification/enrichment step at the end of the procedure adds 58 bases per side to the insert; If the average fragment length of 400 bp refers to the whole library, the insert size would be about 284 bp.

[nucacidhunter]

1- I was thinking to reduce the RNA fragmentation time to 3 or 4 min at 85 deg (down from 5min at 85 deg).

This may not have much effect on library insert size. With TruSeq kit skipping fragmentation increases average size by 50 bp.

- Increase cDNA synthesis time using random hexamers to 60min at 42 deg using AMV (up from 20min at 42 deg)

This may not increase the fragment size either. Reducing random hexamer amount may increase size more, as library fragment size depend on RNA fragment size and number of locations that is primed and extended by RT.

cDNA purification using 0.9x beads (instead off 1.8x previously) - too rough at this step?

This should remove smaller fragments but may cause bias in representation of transcripts in library.

If you are looking to increase insert sizes to avoid sequencing through adapters and maximise useful data, other methods such as oligo dT priming using SMART system followed by cDNA fragmentation or Nextera library prep are better options. In this case one point to note is the aim because the data from large insert will not be easily comparable to previous standard RNAseq data with shorter inserts but it is good approach for studying splice variants and other non-counting applications.

[kerplunk412]

This may not have much effect on library insert size. With TruSeq kit skipping fragmentation increases average size by 50 bp.

This is interesting. Is this something you have done, and if so did you compare complexity, etc. of the resulting libraries? If they were the same it would seem to indicate that cDNA fragment lengths are dictated more by frequency of RT primer binding (or just RT priming maybe) rather than RNA fragment length. So maybe a lower primer concentration could achieve longer cDNAs?

[nucacidhunter]

The figure is based on TruSeq user guide and others experience and libraries were not sequenced. Lowering RT primer concentration expected to increase library fragment size in expense of representation or even coverage of transcripts. This will not be a disadvantage if aim is to study other transcripts features than differential expression.