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/10-290123409_ab1.zip

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

The Geneious files are here (Geneious archive format):

backupordie/lab/Sam/Sequencing_Analysis/Sanger/ireland_clam_RLO/20150309_geneious_Ireland_Clam_RLO.geneious

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:

backupordie/lab/Sam/Sequencing_Analysis/Sanger/ireland_clam_RLO/Clam_RLO_clones_consensus_20150309.fa

Resulting sequence lengths:

SEQUENCE NAME LENGTH (bp)
 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).

Results:

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%

 

EHR vs. EUB

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.

Results:

16s

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

 

EHR

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).

 

EUB

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.

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.

Results:

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 Quantification & PCR – Ireland Clam S/6/14 #19 DNA

Quantification

Quantified the DNA isolated 20150130 via NanoDrop1000 (Thermo Fisher) for quick assessment of DNA.

Although the NanoDrop1000 overestimates actual yields, this is still interesting because the overall yield of this sample is greater than either of the samples isolated on 20150122, yet had significantly less starting material.

 

PCR

Ran PCRs with the following “universal” primer sets in attempt to amplify a 16s (prokaryote) fragment from the RLO that is present in this sample. Additionally, ran a universal 18s (eukaryote) primer set to verify the presence of any amplifiable DNA in the sample, in case none of the 16s primers work.

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

Master mix calcs are here: 20150203 – cPCR Clam debris DNeasy

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,  1x TBE gel w/EtBr

Results:

Ladder is Hyperladder I (Bioline)

Well, ironically, the only thing that shows amplification is the no template controls (NTC) in the universal 16s primer set!  The only useful aspect of this is that it demonstrates that the reagents are functional.

The universal 18s primers don’t seem to amplify anything, either.

Tomorrow, I’ll test these primers out on DNA that I know will amplify, instead of these new clam DNA samples.

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 – Ireland Clam DNA 18s

Since the previous PCR didn’t amplify anything using four different universal 16s (prokaryote) primers, I am testing to verify that the two extractions (QIAamp Fast DNA Stool Mini Kit & the DNeasy Kit; Qiagen) actually isolated any amplifiable DNA using universal 18s (eukaryote) primers.

First, quantified the samples to verify that DNA actually exists in these two samples:

20150128_CgigasOA_BSlibrraryQuants_OluridaLibraryQuants

Sample Concentration (ng/uL)
Clam DNeasy Kit 4.432
Clam Stool Kit 6.184

The yields are surprisingly low, particularly for the DNeasy Kit sample.  In a total elution volume of 200uL, that means I only extracted 800ng…

Due to low DNA concentrations, I used 10uL of each sample in the PCRs.

Master mix calcs are here: 20150129 – cPCR Clam Universal 18s

Samples were run in duplicate.

Cycling params:

  • 1 cycle of 10mins
  • 40 cycles of:
    95C – 15s
    50C – 15s
    72C – 2mins

Results:

Ladder used was Hyperladder I (Bioline).

Neither sample produced any amplification.  The blurry “bands” that correspond to ~100bp are likely RNA carryover from the DNA isolation procedure.  They are not the amplicon we are looking for.  Additionally, they are not primer dimers, as these “bands” do not appear in the NTC.

I believe there is a small quantity of tissue debris in the original EtOH sample tube.  I will attempt to isolate some DNA from this debris and will repeat both the 16s and 18s PCRs.

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

Results:

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.