Sanger Sequencing – pCR2.1/OsHV-1 ORF117 Sequencing Data

Received the Sanger sequencing data back from Genewiz for the samples I submitted last week.

AB1 files were downloaded as a zip file and stored in the Friedman Lab server: backupordie/lab/sequencing_data/Sanger/30-19717124_ab1.zip

Files were analyzed using Geneious 10.2.3.

Geneious analysis was exported (compatible with version 6.0 and up) and saved to the Friedman Lab server:

backupordie/lab/sam/Sequencing_Analysis/Sanger/20170821_oshv_orf117_sanger.geneious

Results:

After vector ID and trimming, all sequences from both colonies were aligned, resulting in an 867bp contig. The size of this contig jives perfectly with the bright PCR band at ~1000bp I saw when screening the two colonies (the ~1000bp includes 300bp of vector sequence from using the M13 primers).

 

The alignment above shows that there were no gaps in the sequencing between the two sequencing primers (M13 forward and M13 reverse). I point this out because the insert in this plasmid was supposed to be the full-length OsHV-1 ORF117 (which is ~1300bp), as described in: Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas. Martenot et al. 2015. As the sequencing shows, that is not what is cloned in this vector.

To determine what was actually cloned in this vector, I performed a BLASTx against the nr database, using the consensus sequence generated from the alignment above:

 

BLASTx generated a total of six matches, five of which match OsHV-1 ORF117 (the hypothetical and RING finger proteins listed above actually have alternate accession numbers that all point to ORF117). However, notice in the one alignment example provided at the bottom of the above image, the Query (i.e. our consensus sequence) only starts aligning at nucleotide 109 and matches up with the NCBI OsHV-1 ORF117 beginning at amino acid 158.

The results clearly show that the insert in this vector is OsHV-1 ORF117, but it is not the entire thing. To confirm this, I aligned the consensus sequence to the OsHV-1 genome (GenBank: AY509253.2) using Geneious:

 

In the image above, I have zoomed into the region in which our sequencing consensus aligned within the OsHV-1 genome. In order to see in more detail, please click on the image above. There are two noticeable things in this alignment:

  1. The insert we sequenced doesn’t span the entire ORF117 coding sequence (the yellow annotation in the image above).

  2. There’s a significant amount of sequence mismatch (112bp; indicated by black hash marks) between the sequenced insert and the OsHV-1 ORF117 genomic sequence from GenBank, at the 5′ end of the insert.

Will pass this info along to Carolyn and Tim to see how they want to proceed.

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 (10-313205054_ab1.zip) 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:

backupordie/Sam/Sequencing_Analysis/Sanger/20151026_RLOv_clones_Sanger_analysis.geneious

Results:

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).

Sanger Sequencing Submission – pCR2.1/RLOv Clones

Submitted the pCR2.1/RLOv clones from earlier this week for Sanger sequencing to Genewiz (order #10-313205054).

Submitted ~500ng of each plasmid in a final volume of 15μL (including primer). Each clone will be sequenced from each direction with M13F (-21) (25pmol; 2.5μL of 10μM stock) and M13R primers (25pmol; 2.5μL of 10μM stock) for a total of 10 sequencing reactions:

  • 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

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/10-291940235_ab1.zip

  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

Sanger Sequencing Submission – pCR2.1/Clam RLO clones

After the previous round of sequencing analysis, decided to sequence a couple of additional clones from each of the three groups: 16s, EHR, EUB. Submitted ~500ng (3μL) of clones #2 & #3 (C2 & C3) from each group to GENEWIZ for Sanger sequencing (Order #: 10-291940235). Each clone was sequenced from each direction with M13F (-21) and M13R primers for a total of 12 sequencing reactions:

  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/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.

Plasmid Isolation – Clam RLO 16s, EHR, & EUB clones

Prepared 1x LB + 50μg/mL ampicillin. Aliquoted 5mL of LB-Amp50 liquid media to 18 15mL conical tubes. Used the restreaked plates created 20150207 and used sterile pipette tips to select each of the six positive colonies from each cloning reaction and inoculate 5mL of LB-Amp50 liquid media. Tubes were incubated O/N @ 37C on a rocker.

All cultures grew. Three milliliters from each culture were used to isolate plasmid DNA using the QIAprep Spin Mini Kit (Qiagen). Samples were eluted with 50μL Buffer EB.

Frozen bacterial stocks were made from each of the six clones, using 500μL of each bacterial culture + 500μL of sterile, 50% glycerol in 2mL screw cap tubes.

Bacterial stocks and plasmid preps were labelled in the following fashion:

  • pCR2.1/Clam RLO 16s C1 – C6
  • pCR2.1/Clam RLO EHR C1 – C6
  • pCR2.1/Clam RLO EUB C1 – C6

where “C#” indicates the clone number. The bacterial stocks were stored @ -80C in the following box “Clones Box 2″:

 

Plasmid preps were quantified on the Roberts Lab NanoDrop1000.

Results:

Spreadsheet: 20150305_ClamRLO_miniprep_ODs

After speaking with Carolyn, she decided she wanted to sequence one clone from each group. Submitted ~500ng of clone #1 (C1) from each group to GENEWIZ for Sanger sequencing (Order #: 10-290123409). Each clone was sequenced from each direction with M13F (-21) and M13R primers for a total of 6 sequencing reactions:

1       SW01    16s_01-M13F(-21)
2       SW02    16s_02-M13R
3       SW03    EHR_01-M13F(-21)
4       SW04    EHR_02-M13R
5       SW05    EUB_01-M13F(-21)
6       SW06    EUB_02-M13R

Sequencing – Coral RLO

Sanger sequencing data from Sonja’s plasmid clones #1, #2, & #3 from 7/29/2013. As of 9/20/2013 Sonja’s notebook is missing, so the full cloning process is not documented. However, I know cloning was done with Invitrogen’s TA Cloning Kit in vector pCR2.1. The three clones were each sequenced with M13 forward and reverse primers. Raw sequence data is here:

20130920_Coral_RLO (Google Drive folder)

  • #1 M13F (039_B10_SJW01-F.ab1)
  • #1 M13R (042_G12_SJW01-R.ab1)
  • #2 M13F (040_A10_SJW02-F.ab1)
  • #2 M13R (043_F12_SJW02-R.ab1)
  • #3 M13F (041_H12_SJW03-F.ab1)
  • #3 M13R (044_E12_SJW03-R.ab1)

Results:

Aligned the sequences to the sequence from which the original primers were designed (Casas et al 2004, Env. Micro). Seqs match nicely, but there are 6 positions (most easily identified in the “Identity” row in the image below) that differ in DQ007350 from our sequencing data of these three clones. The plasmids will be used as a source for qPCR standard curves as well as for in situ hybridization (ISH) probes.