[swbarnes2]

Hello,

Suppose I have two reads from an exome in fastq. How to determine if they are pair-end or single-end or mate-pair ?

Paired end or mate pair reads have to have a means of knowing which two reads go together. Usually, this is by the name. In Illumina at least, reads are normally named by their coordinates on the flowcell. So if you don't have two reads with the same coordiantes, you've got single end.

Paired ends run towards each other, and are about 100-500 bp apart. Mate pairs run away from each other, and tend to be a few kb apart, but I belive sometimes they are contaminated with ordinary paired end data.

[arkal]

Hello,

Suppose I have two reads from an exome in fastq. How to determine if they are pair-end or single-end or mate-pair ?

i could be wrong but i think u can get ur answer by decoding the name of the fastq read... i.e the string following the > symbol.

-A

[raonyguimaraes]

Hello,

Suppose I have two reads from an exome in fastq. How to determine if they are pair-end or single-end or mate-pair ?

[ScottC]

Hi!!
Can anyone clarify the difference between mate pair, pair end and single end reads?

Thanks.

Hi,

Mate-pairs and paired-end reads have been covered. Single end reads are just that... a single read from one end of each sheared DNA fragment.

Scott.

[edilana.gomes]

What is the difference between mate pairs, pair end and single end?

Hi!!
Can anyone clarify the difference between mate pair, pair end and single end reads?

Thanks.

[karve]

This helped but not enough ! I think I get it but then questions pop up - this mates in a pair, paired read stuff pops up when I look a the specification for the SAM format output from the bowtie program. There are flags values that are returned to indicate if the aligned read is one of pair and/or first one in a pair and/or second one in a pair and so on..

But how does it, meaning bowtie, know ? Am I right in thinking that is specified in the input record ? I can see the .fastq input format has a place holder for this, so I'm guessing that other input formats eg. sra also have it ? And if they don't then there's no way for bowtie ( or other algorithms ) to derive it ?

Seeing as this is all equipment specific I'm going to need to look at some videos that describe the front parts of the this entire operation. Any ideas where ? I was hoping to just pick it up and work with it from the point of sequences of characters - an IT perspective - but guess not.

[fedor5002]

About single-end read

For a double-stranded DNA molecule, are the single-end reads generated by sequencing from both 3' ends of the two strands of DNA?

[coswaters]

scaffold and contig helps a lot~ thanks a lot~

[jdrum00]

Thanks to everyone who contributes to these basic threads. I know they may get tedious for the old-timers, but they're vital for newer folks. Even if the same info is technically available at reference sites, it's often easier to understand when the answer is to a specific, basic question, rather than organized as vendor documentation or generalized teaching material!

[krobison]

I am not very familiar with all the biotechnology, so want to ask:
Is it possible to resolve which pair of reads come from the one, unique molecule ? (For instance, that the sequencer tracks this information somehow.)

Yes. In any of these schemes the sequencer keeps track of the physical location in the flowcell of the first read for each polony (spot of DNA) and then

For ligation technologies (Polonator & SOLiD), the reverse reads are really the same technology. For Illumina, there are some clever molecular acrobatics to replace the originally sequenced molecule with it's reverse complement to do paired end sequencing. For mate pairs, I believe they just strip the old extended primer & anneal a new primer. Helicos proposed just adding unlabeled bases for a time period (hence the term "dark fill") and PacBio shuts off their laser and keeps extending (hence "strobe sequencing" is an apropos term).

Complete Genomics used a 4-part read structure & may have gone to even higher number of mates.

Keeping track of the location of polonies or individual DNA molecules is one of the core tricks to all these technologies.

[drio]

I understand that a normal read pair can be aligned with either F-B or B-F orientation. What I'm not too sure about the importance of read1 and read2, does it mean anything if the mapped position of read1 is greater than read2?

Assuming the alignment is correct that may indicate a structural variation happened at that location (you want to check what other alignments on that location tell you).

[hege]

I understand that a normal read pair can be aligned with either F-B or B-F orientation. What I'm not too sure about the importance of read1 and read2, does it mean anything if the mapped position of read1 is greater than read2?

[plichel]

I am not very familiar with all the biotechnology, so want to ask:
Is it possible to resolve which pair of reads come from the one, unique molecule ? (For instance, that the sequencer tracks this information somehow.) I mean usually there are a lot of reads and in diploid systems it might happen there are two 'versions' of the reads.

[krobison]

Mate pair can also cover longer distances & is therefore more sensitive for detecting rearrangements / structural variation.

A third entry in this general category are what Pacific Biosciences calls strobe reads and Helicos calls dark fill. Essentially what happens is that sequence is read then there is a gap of probabilistic length then more sequence is read. This can be repeated many times to give a series of sequence islands separated by gaps with a constrained length distribution.

[flobpf]

I think paired end refers to sequencing from the ENDS of a DNA fragment. If you're doing 75-bp paired end read using an insert size of 300bp, then the machine will sequence 1-75 and 300-225 (for simplicity, omitting the adapters)

Mate pair requires a completely different protocol and is typically over longer distances such as 2-5kb. If you want to sequence a 5kb mate pair library, then 5kb fragments of DNA are isolated on the gel, the ends are biotinylated, the fragment is circularized and sheared. So now when you select using streptavidin, you'll get the fragment that has the ENDS of the original 5kb fragment. This fragment is then sequenced.

Mate pair is more relevant in genome assembly, especially for covering repetitive sequences. Paired end can be used for anything - RNA, DNA.

You can get more information on the Illumina site:

http://www.illumina.com/technology/paired_end_sequencing_assay.ilmn

http://www.illumina.com/technology/mate_pair_sequencing_assay.ilmn

I'm not sure how 454 or other methods define these terms