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Old 04-22-2018, 12:18 AM   #1
soungalo
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Location: Israel

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Cool 454 GS FLX Titanium 'paired end' protocol

Hi,
I know this is an obsolete technology, but I came across some data produced on the 454 instrument (archived in SRA/ENA) which I'd like to use for assembly.
According to the meta data, insert sizes are 8kb and 20kb, but I wasn't able to figure out the protocol used to produce the libraries. I gather that this is some variant on what we'd now call mate-pair, but I'm not sure if I can treat the data exactly the same way as data coming from Illumina mate-pair libraries, in terms of read orientation, insert size distribution etc.
Had anyone worked with such data, or can direct me to some documentation about it? I failed to find anything in the current Roche docs.
Thanks!
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Old 04-23-2018, 09:43 AM   #2
kmcarr
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Quote:
Originally Posted by soungalo View Post
Hi,
I know this is an obsolete technology, but I came across some data produced on the 454 instrument (archived in SRA/ENA) which I'd like to use for assembly.
According to the meta data, insert sizes are 8kb and 20kb, but I wasn't able to figure out the protocol used to produce the libraries. I gather that this is some variant on what we'd now call mate-pair, but I'm not sure if I can treat the data exactly the same way as data coming from Illumina mate-pair libraries, in terms of read orientation, insert size distribution etc.
Had anyone worked with such data, or can direct me to some documentation about it? I failed to find anything in the current Roche docs.
Thanks!

Wow, time to fire up the WayBack Machine Mr. Peabody.

Yes, 454 "Paired End" technology is similar in design to Illumina Mate Pair tech. A large fragment is circularized with a linker, the circular DNA is fragmented and the piece with the linker is enriched. It is then sequenced and the linker sequence is identified to separate the "forward" and "reverse" reads. I have attached a 454 data processing manual; look at section 4.6 for information about paired end data.

Here are some links to previous thread on SeqAnswers discussing 454 paired end reads:

http://seqanswers.com/forums/showthread.php?t=8677
http://seqanswers.com/forums/showthread.php?t=12940

This latter thread includes a post with the linker sequences (post #2). There are different linker sequencing depending which generation of the library prep kit was used, FLX or Titanium.
Attached Files
File Type: pdf USM-00058.09_454SeqSys_SWManual-v2.9_PartC.pdf (5.24 MB, 1 views)
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