Hi,
I think I am having trouble understanding PCR and trimming tools. After amplification using forward and reverse primers in PCR, wouldn't the resulting strands include forward primer, reverse primer, reverse-complementary forward primer and reverse-complementary reverse primer?
from the original antisense DNA strand (3' -> 5') for instance, PCR will result in:
5' forward primer ..... DNA sequences .... complementary of reverse primer 3'
3' complementary forward primer .... complementary DNA sequences .... reverse primer 5'
I have got reads from PacBio (single-end library), and I assume the sequences are from 5' to 3'. Then, does this mean that the amplified 3'->5' strand will be reverse in the reads, resulting in the reverse primers too? For instance, complementary forward primer -> reverse-complementary forward primer?
If I were to trim the forward primer using a software, will this also detect complementary forward primer and reverse-complementary forward primer sequences?
Thank you in advance!!
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written
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s.kyungyong64 •
1 answer
1) SMRT sequencing of PCR amplicons will yield sequences of both + & - strands (for raw reads as well as for ccs reads).
2) Whether primer sequence in both the strands will be trimmed depends on what approach/tool you are using to do this. Are you trimming the reads based on a fixed length of 70 bases or after searching for the primer sequences? Both these approaches have limitations because we have seen that the SMRT reads from PCR amplicons may be truncated and also has greater noise towards both the ends.
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Are these CCS reads? How long are they, and how long are the primers? Also, what kind of data is this?
Also, to clarify, DNA sequences are "forward" or "reverse-complement". They are never (in normal situations) just "reverse" or "complement".
This is Pacbio sequencing data, and I believe these are CCS reads. Primers are about 70 nucleotide. And yes, you are right! I have been confused!