Targeted transcript sequencing-Iso-Seq

[Fad2012]

Hello guys,

Hi Guys,

I am preparing to use PacBio Isoforms sequencing, to sequence the total human RNA searching for specific transcripts, which are 7.4-7.7kb long, and occurring in a very low frequency (~1%, if not lower) among total RNA. The main goal is to characterise and quantify these specific, low-frequency RNA molecules. I want to kindly ask for your advice as I am not sure of the following points:

1) How deep could the PacBio Iso-Seq detect in terms of number of transcript? I have been searching in vein to find a reference for this!

2) Will it help to reliably detect those rare molecules if I increase the number of SMRTcells?

3) Taking into consideration the length of the target transcripts (7.4-7.7kb), how many full transcript could I retrieve with a reasonable number of passes, say around >=3, in other words, how good is the chance for at least 1/3 of ZMW productivity 1 to get a high quality Read of Insert?

Many thanks

[tysonaclark]

I can answer most of these questions:

1) With the current Iso-Seq workflow (and a properly loaded sample), on a per-SMRT Cell basis, you should get ~50,000 reads with ~20,000-25,000 being full length reads.

2) Yes, increasing the number of SMRT Cells will improve your chances of seeing rare transcripts / rare isoforms. It's a numbers game and depends on how abundant the transcript is.

3) With the current sequencing chemistry (P6/C4), the average read length is ~10-15 kb. There is a distribution of lengths and I don't know off the top of my head how many will provide 3+ passes on a 7kb insert. However, I'm not sure that 3+ passes will be necessary to answer your experimental questions.

Have you considered doing targeted Iso-Seq? It might work well for this project.

- Tyson Clark

[Fad2012]

Hi Tyson,

I thought the 3 passes will provide enough data for building the correcting the sequencing error. However, it seems that I did not take into consideration the new sequencing chemistry. Is there any source that can help me to understand the error correction of the long Reads of Insert (in my case 7.5kb), and how one pass of the polymerase will guarantee a reliable read?

Many thanks