Dear All,
I am currently working with a RAD sequencing data set, sequenced on a PE-150bp Hiseq and I am now wanting to assemble my data into wonderful and long contigs (all cleaned and de-multiplexed and ready to go!).
The standard that appears to be used with RAD sequencing assembly is velvet, so I want to start with that but I am happy to hear about other people's experiences or recommendations for other software?
I also have a question regarding simple Illumina paired-end sequencing, because I am afraid I am a bit confused.
"Read 1 is generated using the READ 1 Sequencing primer. The read 1 product is removed and the Index Sequencing Primer is annealed to the same strand (...). If a paired-end read is required, the original strand is used to regenerate the complementary strand. Then, the original strand is removed and the complementary stand acts as a template for application read 2."
So basically, on the Illumina machine, a complementary strand to Read 1 is created but not in that sense read or sequenced. And then this complementary and non-read strand acts as a template for read 2 (as the sequencing reaction always occurs 5' to 3'), so basically Read 1 and Read 2 are NOT reverse complementary to one another? Is this correct? There seem to be some posts and blogs that state that the forward and reverse reads are reverse-complementary to each other in orientation!
Many thanks in advance!!
Sarah
I am currently working with a RAD sequencing data set, sequenced on a PE-150bp Hiseq and I am now wanting to assemble my data into wonderful and long contigs (all cleaned and de-multiplexed and ready to go!).
The standard that appears to be used with RAD sequencing assembly is velvet, so I want to start with that but I am happy to hear about other people's experiences or recommendations for other software?
I also have a question regarding simple Illumina paired-end sequencing, because I am afraid I am a bit confused.
"Read 1 is generated using the READ 1 Sequencing primer. The read 1 product is removed and the Index Sequencing Primer is annealed to the same strand (...). If a paired-end read is required, the original strand is used to regenerate the complementary strand. Then, the original strand is removed and the complementary stand acts as a template for application read 2."
So basically, on the Illumina machine, a complementary strand to Read 1 is created but not in that sense read or sequenced. And then this complementary and non-read strand acts as a template for read 2 (as the sequencing reaction always occurs 5' to 3'), so basically Read 1 and Read 2 are NOT reverse complementary to one another? Is this correct? There seem to be some posts and blogs that state that the forward and reverse reads are reverse-complementary to each other in orientation!
Many thanks in advance!!
Sarah
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