Quick Start Guide

Submitting DNA samples For Bi-Directional Sequencing

In this segment of the DNA barcoding workflow, you will submit COI amplicons generated in your science lab to a sequencing facility for bi-directional sequencing (i.e. sequencing the sense and antisense strands of each COI amplicon). Because the reverse complement of the nucleotide sequence generated for one strand matches the nucleotide sequence generated for the other (except in non-overlapping regions), bi-directional sequencing essentially doubles the amount of sequence data available for each amplicon. As discussed in greater detail below, this duplication of information can help resolve ambiguities in nucleotide sequence data that arise from technical limitations of dye terminator cycle sequencing.
Most DNA sequencing facilities make use of online submission forms that require you to provide the following types of information for each DNA sample:

1. The format in which DNA samples are delivered (e.g. in tubes or multi-well plates)
   To avoid sample mix-ups, we strongly recommend that you submit DNA samples in 0.5 mL (or smaller) tubes that are clearly labeled with an appropriate sample ID (see below for additional details).
2. The method of payment (e.g. purchase order number, credit card number, etc.) Most schools and universities will require you to obtain a purchase order (PO) number from an authorized purchasing agent.
3. The type of DNA sample being submitted (e.g. plasmid DNA, PCR product, etc.) Indicate that you are submitting samples containing PCR product.
4. The type of DNA sequencing service requested
   At the conclusion of the initial PCR reaction used to generate COI amplicons, each reaction tube contained unincorporated nucleotides and primers, Taq polymerase, and magnesium. The presence of these reaction components will adversely affect the sequencing reaction and consequently produce low quality or uninterpretable sequence data. It is therefore imperative that you purify COI amplicons prior to submitting them to a sequencing facility. If your COI samples were purified, then you should request standard sequencing. If not, then you may request this service for an additional charge. Please be advised that some sequencing facilities do not offer this service to its clients.
5. The method used to purify the DNA
   If applicable, indicate the name of the vendor that supplies the reagents that your students utilized to purify each PCR reaction.
6. The name or ID of the DNA sample
   The ID of the DNA sample should match the ID of the corresponding tissue sample from which it was obtained. We strongly discourage the practice of re- coding DNA samples as it increases the likelihood of sample mix-ups. Ensure that the side or lid of each sample tube is clearly labeled with the appropriate ID. Since COI amplicons will be sequenced bi-directionally, you should aliquot or apportion an equal volume of each DNA sample into two separate tubes (unless you are otherwise instructed by the sequencing facility). For a purified DNA sample with the ID code CMB12, one volume should be aliquoted into a tube labeled CMB12-F and an equal volume should be aliquoted into a tube labeled CMB12-R. The forward sequencing reaction will be performed using template DNA in the tube labeled CMB12-F, and the reverse sequencing reaction will be performed using template DNA in the tube labeled CMB12-R. Both codes should be entered into the sample ID column of the online submission form. You should also specify on the form that the forward sequencing primer should be used for sample CMB12-F and that the reverse sequencing primer should be used for sample CMB12-R.
7. The name of the sequencing primer
   Your choice of sequencing primers will be dictated by the primers that your students used to generate COI amplicons during the initial PCR reaction. iBOL researchers sometimes use M13-tailed PCR primers, which contain at their 5’ ends a short string of nucleotides derived from M13 DNA. These short nucleotide sequences of are viral in origin and therefore not represented in the COI template. Downstream of the M13 sequences, the primers also contain a continuous series of nucleotides that are complementary to those flanking the barcode region of the COI gene. During the annealing step of PCR, these complementary primer sequences bind to the sense and antisense strands of the COI gene according to the base pairing rules, allowing Taq polymerase to copy the intervening region of DNA during the subsequent elongation step.
   Importantly, the M13 region of each primer cannot bind the template DNA during the annealing steps of PCR (because complementary nucleotides are not present in the COI gene). However, during the subsequent elongation step, these short M13 nucleotide sequences are incorporated into the 5’ ends of each newly synthesized strand of DNA along with the remainder of each primer sequence. The COI amplicons generated during PCR therefore contain different M13 nucleotide sequences at each end of the sense and antisense strands. These M13 nucleotide sequences serve as binding sites for universal sequencing primers used by most sequencing facilities. If you used M13-tailed PCR primers, the universal M13F-20 primer can be selected for the forward sequencing reaction of each COI amplicon, and the universal M13R primer can be selected for the reverse sequencing reaction of each COI amplicon.
   
   If your PCR primers lack M13 tails, then the forward PCR primer can be selected for the forward sequencing primer and the reverse PCR primer can be selected for the reverse sequence primer. In this case, you will need to submit an aliquot of each primer to the sequencing facility and provide the sequence of each primer with your submission.
8. The length (in bp) of the DNA sample to be sequenced
   The standard length of the COI barcode region is ~650 bp.
9. The concentration of the DNA sample (in ng/µL)
   In order to generate high quality sequence data, sequencing facilities generally require a DNA concentration in the range of ~5-20 ng/µL. The DNA concentration of each sample can be determined using a spectrophotometer or by running an agarose gel to compare the band intensity of a small volume of sample DNA with the band intensity of DNA concentration standards. Since standard spectrophotometers found in most educational science labs yield highly variable and inaccurate values for PCR products, the latter method is recommended.
10. The total amount of DNA sample
    This value is determined by multiplying the DNA concentration of the sample by the volume (in µL aliquoted into a given sample tube. In general, sequencing facilities recommend that you submit a total of ~100 ng DNA. This amount of DNA enables the sequencing facility to repeat a sequencing reaction in the event that a procedural error occurred.

The section above is intended to provide you with a basic understanding of the types of information that a sequencing facility will request. Before submitting your DNA, we strongly encourage you to contact the sequencing facility specific submission guidelines and procedures.