Sanger Sequencing Data – pCR2.1/Clam RLO clones

Received data from yesterday’s sequencing submission for GENEWIZ order: 10-291940235.  Clones from each of the three groups (16s, EHR, EUB) were sequenced (see below).

Raw sequencing data (.ab1) files were stored on: backupordie/Sequencing Data/Sanger/

  1. SW01 16s_C2_01-M13F(-21)
  2. SW02 16s_C2_02-M13R
  3. SW03 16s_C3_01-M13F(-21)
  4. SW04 16s_C3_02-M13R
  5. SW05 EHR_C2_01-M13F(-21)
  6. SW06 EHR_C2_02-M13R
  7. SW07 EHR_C3_01-M13F(-21)
  8. SW08 EHR_C3_02-M13R
  9. SW09 EUB_C2_01-M13F(-21)
  10. SW10 EUB_C2_02-M13R
  11. SW11 EUB_C3_01-M13F(-21)
  12. SW12 EUB_C3_02-M13R

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.

Colony PCRs – Clam RLO 16s, EHR, EUB

Colony PCRs were performed on each of the three transformations from yesterday (16s, EHR, and EUB primers) using the M13F/R vector primers. Colonies were picked form the transformation plates with pipette tips, re-streaked on a secondary, gridded, numbered LBAmp50+x-gal plate and then used to inoculate the respective PCR reactions. Six white colonies (positive clones) and a single blue colony (negative clone) were selected from each transformation.

Restreaked plates were incubated @ 37C O/N and then stored @ 4C (Parafilmed).

Master mix calcs are here: 20150227 – Colony PCR Clam RLO

30μL of each reaction was run on a 1% agarose 1x Low TAE gel, stained w/EtBr.


Ladder: Hyperladder I (Bioline)

Upper Left: 16s colonies 1 – 7

Upper Right: EHR colonies 1 – 6

Lower Left: EUB colonies 1 – 7

Based on the PCRs used for cloning, all white colonies screened exhibit the expected product sizes. Additionally, each of the blue (negative) colonies, produced the expected band size that are indicative of an empty plasmid.

Will select a positive colony from each set for mini prep and Sanger sequencing.

PCR – Ireland Clam RLO DNA S/6/14 #19

This is an exact repeat of the PCR from yesterday, but with a brand new vial of Apex Red Master Mix, in an attempt to eliminate the contamination previously seen in the NTCs.


Ladder: Hyperladder I (Bioline)

Well, for some reason there are still bands in the NTCs. However, they appear to be of different sizes than the bands in the clam DNA samples. I think they’re OK to use and the cloning/sequencing is cheap enough these days, that I’ll just get these sequenced and see what we have.

I excised each of the bands in the clam DNA samples (16s = ~2000bp; EUB = ~2100bp) and purified them using Ultrafree-DA spin columns (Millipore) in preparation for cloning.

PCR – Ireland Clam RLO DNA S/6/14 #19 from 20150130

After the last PCR continued to exhibit products in the no template controls (NTC) for most of the primer sets I was using, I ordered new primers. They arrived today so, I re-ran the PCR on the clam RLO DNA isolated 20150130 with the following new primers:

Master mix calcs are here: 20150223 – cPCR Universal Primers Apex Red MM

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 1min

Samples were run on 1.0% agarose, low TAE gel stained w/EtBr.


Ladder: Hyperladder I (Bioline).

Crazy; contamination still present in the NTCs. Primer stocks were steriley reconstituted with Low TE Buffer (IDT) and working stocks were created steriley, so I’m not really sure why this is continuing to happen. Possibly the polymerase is contaminated?  Will try again with previously unopened polymerase and see how that plays out.

No bands were excised since I can’t be certain that the bands present in the Clam DNA samples are from the actual sample and not from the apparent contamination.

PCR – Ireland Clam RLO DNA S/6/14 #19 (from 20150130)

After previously confirming that the issue with previous PCRs was due to bad reagents, I re-ran the PCR on the clam RLO DNA isolated 20150130 using a set of universal 16s primers, as well as a universal 18s primer set to serve as a positive control that amplifiable DNA was present in the sample.

Master mix calcs are here: 20150219 – cPCR Universal Primers Apex Red MM

Primers being used are:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 18s EUK 581 F, 18s EUK 1134 R

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 1mins

Samples were run on 1.0% agarose, low TAE gel stained w/EtBr.


Ladder used was O’GenRuler 100bp DNA Ladder (Thermo-Fisher).

No sample was loaded directly next to ladder to facilitate excision, if necessary.

Each sample was accompanied by a no template control (NTC).

The ehrlichia universal primers (EHR) and the universal 18s (18s) primers are the only two primer sets that do not have contamination present in the NTCs.

Excised the EHR band and purified with Ultrafree-DA columns (Millipore). Purified DNA was stored @ -20C and will be used for cloning/sequencing next week.

Have already ordered additional primer sets of those above that are contaminated. Will re-run the PCR with those new, sterile primer sets when they arrive to obtain a larger product (the EHR amplicon is only ~350bp).

DNA Isolation – Ireland Clam Sample S/6/14 #19

Previous attempts at isolating usable DNA failed.  Previously used both the QIAamp Fast DNA Stool Kit and DNeasy Kit (Qiagen) and both yielded nothing. In a last ditch effort, since there’s no tissue left, I pellet the remaining tissue/debris, removed the EtOH and processed the sample with the DNeasy Kit (Qiagen), following the animal tissues spin protocol.  DNA was eluted with 100uL of Buffer AE.

Will quantify and PCR next week.

PCR – Universal 16s in Ireland Clam DNA

In an attempt to identify a potential rickettsia-like organism (RLO) in the clam sample, ran PCRs with various universal 16s primers:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B

Template DNA used were the Ireland clam DNA isolated yesterday/today with the Qiagen stool/DNeasy kits, respectively.

Master mix calcs are here: 20150122 – cPCR 20150122 – cPCR Clam Universal 16s

All samples were run in duplicate.

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 2mins

Ran samples from yesterday’s PCR out on a 0.8% agarose, low 1x TAE gel w/EtBr


No amplification in either sample (stool or DNeasy kit DNA) with any of the four primer sets.  Will discuss with Carolyn how she wants to proceed.


DNA Isolation – Ireland Clam Tissue

Performed DNA isolation on clam tissue (sample: S/6/14 #19) supplied by Deborah Cheslett in July 2014 (based on date on letter accompanying the sample).  Sample was preserved in 80% EtOH.  Isolated DNA using the QIAamp Fast DNA Stool Kit (Qiagen), per Carolyn’s request.

Removed tissue from EtOH, blotted dry with Kim Wipes.  Tissue type was not noted in the letter accompanying the sample.

Tissue weighed 117mg, which is just below the recommended range for the the Qiagen stool kit (180 – 220mg is recommended).  Minced tissue with razor blade and processed according to the manufacturer’s protocol for pathogen detection.  Because tissue was very dense/rubbery, the tissue did not lyse during the initial incubation period (95C for 5mins).  Extended this incubation to 2.5hrs in an attempt to lyse the tissue.  Lysis did not fully dissolve the tissue, which was not surprising.

Proceeded with the manufacturer’s protocol and eluted with 100μL of Buffer ATE.

Retained remaining supernatant from the lysis step.

Processed the remaining unlysed tissue using the DNeasy Blood & Tissue Kit (Qiagen) according the manufacturer’s protocol.  Tissue lysis step was performed at 56C O/N.

Sample was eluted with 200μL of Buffer AE.