Sanger Sequencing Analysis – pCR2.1/RLOv Clones

Sequencing results from the samples that were submitted to Genewiz on Friday have come back:

  • SW01    RLOv_DNA_Helicase-M13F_-21_
  • SW02    RLOv_head_to_tail-M13F_-21_
  • SW03    RLOv_membrane_gene_1-M13F_-21_
  • SW04    RLOv_membrane_gene_2-M13F_-21_
  • SW05    RLOv_tail_fiber-M13F_-21_
  • SW06    RLOv_DNA_Helicase-M13R
  • SW07    RLOv_head_to_tail-M13R
  • SW08    RLOv_membrane_gene_1-M13R
  • SW09    RLOv_membrane_gene_2-M13R
  • SW10    RLOv_tail_fiber-M13R

The data ( has been stored in the following location: backupordie/sequencing_data/Sanger.

Sequences were loaded into Geneious (v.9.0.2). Vector sequences were trimmed/annotated using the Trim Ends with UniVec feature in Geneious.

Each clone was sequenced once from each direction, so the two sequences generated from each clone were mapped to the original sequence from which the primers were designed using Geneious Mapper.

The Geneious analysis was exported and saved in the following location:



Each clone’s sequence matches that of the source sequence, so we’re good to go!

Will proceed with dye-based quantification of each plasmid. Will then proceed with developing ISH probes (membrane genes 1 & 2, tail fiber gene) or qPCR standard curves (DNA helicase, head-to-tail).

In the alignments below, the reference sequence is highlighted in light yellow. The two electropherograms are align below the reference. The grey line in the consensus sequence indicates any sequence disagreements by placement of a black mark at the position. However, the sequences all match, so there are no black marks in the regions between the identified vector sequences (red annotations below each electropherogram).

Sequence Data Analysis – pCR2.1/Clam RLO 16s, EHR, EUB

Sequencing data was received back from GENEWIZ on Friday. The ZIP file containing all six sequence trace files (.ab1) was moved to the lab server:

backupordie/lab/Sequencing Data/Sanger/

The data files were copied to Geneious (v.7.1.7; Biomatters Ltd.) for initial manipulation.

The Geneious files are here (Geneious archive format):


Quality trimming and vector sequence identification was performed.
All trimmed pairs of files were aligned using the built-in Geneious aligner. Default settings were used, except “Automatically determine sequence direction” box was checked. The alignments were visually inspected for mis-called bases and corrected where necessary. The resulting consensus sequences from each clone were exported to separate files, as well as a single, multi-FASTA file:


Resulting sequence lengths:

 16s_consensus_sequence  1507
 EHR_consensus_sequence  198
 EUB_consensus_sequence 1532

These consensus sequences were aligned to each other using the MUSCLE alignment in Geneious, using default settings (click on images below to enlarge).


The alignments below show two things:

  1. Similarity (identity) between the sequences being aligned. This is represented as the green bar(s) above the alignments. The more green, the more sequence identity is shared between the two sequences.

  2. The alignments between the two sequences are represented as black bars next to the corresponding sequence name. A black bar/box indicates exact sequence matches between the two sequences. A black line is indicates region(s) where the sequences do not match.


16s vs. EHR

Similarity: 11.25%


16s vs. EUB

Similarity: 85.18%



Similarity: 12.37%


The EHR sequence shares little similarity to the other two sequences.

The 16s & EUB sequences are highly similar, but not identical.


Each of the three sequences (using the multi-FASTA file referenced above) was BLAST’d (blastn) against the NCBI nr database.



The sequence produced using the 16s primers is clearly amplifying the 16s sequence of Vibrio tapetis, a pathogen of cultured clams.



The sequenced captured by the EHR primers has no matches at all in the NCBI nr database. This is likely due to the length of the sequence (only 198bp), however, it’s still long enough that I feel it should match something. Also, just putting this here as a reminder, the EHR primer set is the only set that didn’t produce amplification in the no template controls (NTC).



The product of the EUB primers matches very well to the 16s sequence of a variety of uncultured bacteria species.


I will relay the results to Carolyn and see how she’d like to proceed. Due to the nature of what’s being done here (using universal 16s bacterial primers), I think it would be good to sequence additional clones from each of the three cloning reactions to see if we pick up additional sequences.