Do paired end reads come from opposite strands or the same one?

[AmitL]

Thanks in advance,
Amit

[Brian Bushnell]

In a normal Illumina or Solid fragment library, opposite strands. But particularly with long-mate-pair protocols, the answer may be different, so really you should ask whoever generated the data.

[Chipper]

Opposite strand for Illumina, same strand for SOLiD (if they still use the original method).

[AmitL]

Thank you very much, Brian and Chipper.

[volks]

not sure if this is the question .. for illumina sequencing they actually both come from the same strand, just one of them shows the reverse complement (have a look at the details of the protocol). this is for example important for bisulfite sequencing.

[Brian Bushnell]

Actually, read 2 is synthesized on the opposite strand for Illumina (the enzyme goes forward for both reads so they need opposite template strands), and the same strand in Solid (a different enzyme is used for read 2, which goes backwards). Though in terms of Illumina bisulfite, I suppose the treatment would be done when it was single-stranded and thus be done on the same strand for read 1 and 2, but I've never done bisulfite experiments so I'm not sure.

When you are mapping or assembling Illumina or Solid fragment libraries, read 1 and read 2 will end up on opposite strands.

[dpryan]

Well, they both originate from the same original strand but will normally produce sequence for both (so read #1 might produce that of the + strand and read #2 that of the - strand). I say normally because this isn't the case in bisulfite sequencing, for example, where if read #1 gives sequence for the + strand then read #2 will not give the sequence for the - strand, due to the two strands not being complementary.

[Jayakumar S]

Actually, read 2 is synthesized on the opposite strand for Illumina (the enzyme goes forward for both reads so they need opposite template strands), and the same strand in Solid (a different enzyme is used for read 2, which goes backwards). Though in terms of Illumina bisulfite, I suppose the treatment would be done when it was single-stranded and thus be done on the same strand for read 1 and 2, but I've never done bisulfite experiments so I'm not sure.

When you are mapping or assembling Illumina or Solid fragment libraries, read 1 and read 2 will end up on opposite strands.

Hi,
I am having a basic question.
In WGS, where we will have millions of reads, how we can identify the sequences of positive and negative strands of a single fragment?.
Is it is based on the adapter sequences attached to it?. If so, we will have only few adapters for the millions of reads?.
Please clarify.

Jayakumar S

[dpryan]

You align it and look at the orientation of read #1. Depending on the nature of the library prep (i.e., whether it was stranded and, if so, which type), that will tell you. There's generally no a priori way to otherwise know.

[Jayakumar S]

You align it and look at the orientation of read #1. Depending on the nature of the library prep (i.e., whether it was stranded and, if so, which type), that will tell you. There's generally no a priori way to otherwise know.

Thanks Ryan for your response.
Got a fair idea about it. But, still not getting a clear picture.
The read1 and read2 orientation will be in the opposite direction, whereas the PCR duplicates will be in the same direction as read1.
Please correct, if I am wrong.

Thanks
Jayakumar S

[dpryan]

PCR duplicates may or may not be in the same orientation after sequencing. This will be dependent upon the type of library prep.

[Jayakumar S]

PCR duplicates may or may not be in the same orientation after sequencing. This will be dependent upon the type of library prep.

Thanks Ryan.
Then, during alignment process, for the read2, whether the software will take the complementary sequence?.

[dpryan]

Most any modern aligner (unless they're intended for other purposes) will attempt to align reads in each orientation and then match them properly given the library type. This isn't something you need to worry about unless you're writing an aligner or are getting strange results.

[Jayakumar S]

Most any modern aligner (unless they're intended for other purposes) will attempt to align reads in each orientation and then match them properly given the library type. This isn't something you need to worry about unless you're writing an aligner or are getting strange results.

Thanks Ryan for all the clarification.

Jayakumar S