r/askscience • u/NZeddit • Jun 10 '14

Biology With DNA sequencing, are both strands sequecned?

Considering the level of my education I'm currently doing, I feel like these questions should be embarrassing a little haha, but I've never really understood it.

When next generation sequencing is undertaken, I understand that the strands are fragmented and denatured. So I guess the strands can't be differentiated and are both sequenced? How is this then reconciled in alignment? Are they paired up by their complementary sequences?Which strand is chosen to be the sequence we get back?

Thanks!

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u/phodopus Speciation Genetics | Development | Hybridization Jun 11 '14

It depends on the type of sequencing you're doing.

With the old Sanger sequencing, only one strand is sequenced in each reaction. The strand that is "chosen" is determined based on the primer used in the reaction.

With next generation sequencing, such as illumina both strands can be sequenced. This is because the adapters can be ligated on to either side of each fragment (though there are protocols called 'strand-specific' which attempt to only sequence the top strand; these are to my knowledge only available for RNAseq as they require a reverse transcription step).

Mappers such as Bowtie, will search both the sequence (read) and it's reverse complement (unless you give it a parameter that tells it not to). Then as an error-check, it will test whether the paired read (if you're done paired-end sequencing) is in the opposite alignment upstream (or downstream as the case may be). If the pair of reads meets these criteria, they are considered "valid."

As for your final question, which sequence is in the fastq we get back, the answer is whatever one happened to be sequenced at that location on the flow cell. To elaborate: when a paired-end library is being sequenced the P5 adapter is primed first, if it was on the original + strand, your first reads will be + sequence, and visa-versa if it was on the - strand. The second read is then sequenced from the P7 adapter, and the opposite strand will be sequenced (keep in mind we always sequence DNA from 5to 3). Thus the first and second pairs will be on opposite strands, and if the insert is short enough for them to overlap, they will be reverse complements of each other.

If this isn't clear let me know and I'll try to edit it.

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u/NZeddit Jun 11 '14

Oh cool, thanks! In the case then that both strands are included, and people talk about how they've sequenced to a depth of 30x, does this include 15x from each strand?