# AMR Genomics Meetings
## Write down your updates on:
* What you worked on the past 2 weeks
* The challenges you have faced
* What is scheduled for the next 2 weeks

## February 27 2024
Sign in:
1. James Wachira
2. Brian Kimutai

### Brian Kimutai
_Updates_
* GREATLIFE Scoping review
-Completed data analysis of 210 articles included in the review, and Have began drafting the manuscript.
* Analysed DHIS2 data for GREATLIFE Project sites selection. 7 Counties with high diarrhea rates and diverse ecological conditions were considered. Dispensaries (level 2) and health centers (Level 3) at the frontline will be considered.
* Reviewed the KASH conference report by Latiffah Benta

_Plan_
* To continue with drafting the Scoping Review Manuscript
* To Map out the precise location of health facilities and rural Villages that will serve as sites for the GREATLIFE project
* To attend The GREATLIFE Strategy Meeting On 18-03-2024.

## January 30 2024
Sign in:
1. James Wachira
2. Brian Kimutai
3. Vanessa Natasha
4. Latifah Benta
5. Collins Kigen

### James Wachira
_Updates_
* Performed serotyping of the Shigella and E.coli genomes for the EID MHK project
* Took part in the PDP 2 library prep and sequencing alongside the genomics team
* Basecalling using dorado performed for the PDP2 pod5 files awaiting genomic characterization

_Challenges_
* Performing in-silico pathotyping of E.coli using WGS data. This is mainly done by PCR and there exists quite several updates to the virulence gene alleles used in differentiating the different pathotypes of E.coli
* Delays in sequencing due to low count in number of available pores on 2 FLOMIN114 flow cells

_Plan_
* Work on KASH presentation slides
* PDP2 genomic characterization as well as report generation

### Brian Kimutai
_Updates_
* GREATLIFE Scoping review
-Completed data extraction, a total of 210 study articles were analysed.We have also began data synthesis which involves Summarizing and analyzing the extracted data to identify key themes, trends, and patterns across the literature.
* Worked on GREATLIFE Project SOPs,
* KASH abstract - Began developing presentation slides for the KASH conference.

_Challenges_
* Occasional network downtime- Conducting data extraction and synthesis requires internet access, the occasional network fluctuation would really hamper progress.

### Vanessa Natasha
_Updates_
* Created relative abundance plots for the EID manuscript(Relative abundance of AMR, plasmids, and virulence factors)
* Was part of the library prep and sequencing of the PDP2 samples alongside the genomics teams
* Performed statistical significance test between sample type and strain type in the EID work
* Wrote the initial PDP2 report

_Challenges_
* Statistical analysis calculation using categorical data
* Sequencing challenges due to the low number of pores in the available flow cells

_Plan_

* Completion of KASH slides
* EID MHK analysis report
* EID MHK visualizations
* PDP2 sample characterization

### Latifah Benta
_Updates_
* Worked on SOPs that will be used for the GREATLIFE project._
* Finnished extraction on the articles for GREATLIFE (Extracted 65 articles)
* Prepared wet slides for Parasitology and observed _Cryptosporidium spp._, _giardia_, under the microscope guided by Janet and Ronald
* Cultured bacterial isolates with Ondolo to test Vitek
* Worked on addressing KASH reviews
* Loaded samples for sequencing guided by Vanessa and James.

_Challenges_
*N/A

_Plan_
* Start on synthesis and working on a draft manuscript for the GREATLIFE Project.
* Work on Slides for the KASH presentation
*

### Collins Kigen
_Updates_
* EID Supplement manuscripts - Performed bioinformatics analysis for the 2 manuscripts from AMR and Enterics.
* Pathogen Discovery Project 2 - PArticipated in library preparation, sequencing, bioinformatics analysis and report writing for PDP2 samples
* Installation of genomics equipment - Coordinated the plans to install the NextSeq 1000
* Greatlife nanopore training - Lead the techical team for the nanopore training. So far, outlined the trainign objectives and list of equipment and reagents for procurement.
* Wellcome Connecting Science AMR course - Particpated in the planning and drfating of manual for the WCS AMR course
* WRAIR training visit - Outlined training objectives and schedule.
* Manuscripts review - Reviewed E. coli Abstract for ASM conference
* Our paper for The first report of mcr-10 in Kenya was published online
_Challenges_
_Plan_

### Consolidated Updates
#### AMR (CK)
* EID-AMR
* EID-MHK
* PDP2
* mcr-10 manuscript

## January 16 2024
Sign in:
1. Vanessa Natasha
2. Brian Kimutai

### Vanessa Natasha
_Updates_
* Created heatmaps for the EID manuscript
* Created alluvial diagrams for the EID manuscript
* Intial visualizations for KASH abstract that will be used in presentation

_Challenges_
N/A

_Plan_

* Complete the pending visulaizations of the EID manuscript
* Complete KASH visualizations

### Brian Kimutai
_Updates_
* Greatlife project;
-Attended Greatlife Project Collaborative meeting on Jan 11, 2024 10:00 AM.Gave progress report of the Scoping review.
* Greatlife Scoping Review - Did data extraction of 30 study articles
* Parasitology
-Worked with Janet to prepare wet mounts and did acid-fast staining (Modified Kinyoun acid fast staining ) for identification of parasites in concentrated stool samples.
* Bacteriology
-Was guided on various media used at MHK for identification of pathogenic bacterial species in stool samples.

_Plan_
* Finish up on data extraction and begin data synthesis,
* Prepare KASH presentation slides,

## November 6 2023
Sign in:
1. James Wachira
2. Vanessa Natasha
3. Brian Kimutai
4. Latifah Benta
5. Collins Kigen

### James Wachira
* Library prep and sequencing of Enterococcus phages on Illumina Miseq
* Performed a fastqc on E,faecium raw reads. Reads are good quality (in the Q30 quality score region
* Took part in compiling a script for runnning the E.coli genomic analysis for the EID project
* Curation of the assembly data in readiness for bioinformatic analysis for the EID project
* E_coli analysis started :- MLST, Phylogroup typing, Serotyping, AMR and Virulence factor screening
* Attended a webinar on "A new age in liquid biopsy research with highly accurate short-read sequencing" organized by PACBIO. The talk was centered on detecting cancer by sequencing "cell-free RNA" in a patient's blood to test for the presence of both protein-coding and repetitive noncoding RNA. This is made possible by PacBio's short-read sequencing-by-binding (SBB) chemistry
* Receipt of the PDP samples in readiness for the blind exercise

_Challenges_
* Addition of a new component to the E_coli analysis workflow: Fimtyping. A tachnical issue with running the program but eventually managed. Need for test data to validate the tool

_Plan_
* Completion of the PDP exercise
* Klebsiella pneumoniae workflow development

### Vanessa Natasha
_Updates_
* Partcipated in library prep for Enetrococcus phages on the Illumina Miseq platform
* Completed E.coli visulaization
* Tested a piepline for Insertion Sequence screening for the _Acinetobacter baumannii_ sequences on PanISa and ISFinder
* Made a presentation for Journal club
* Instructed a class on MLST during the Bioinformatics trainning
* Assisted in the curation of an inventory list of molecular reagents to be transported to Kericho
* Involved in script compiling for the EID analysis
* Receiving PDP samples and assisting in drafting the initial receipt report

_Challenges_
* Failing to install MGE due to a bowtie2 error
* Running panISa and debugging the pipeline error

_Plan_
* Complete screening of insertion sequences on A.baumannii sequences
* Initial visualization of EID analysis
* Final KASH abstract draft
* PDP sequencing and analysis

### Brian Kimutai
* Bioinformatics Training-
-Was introduced to strain typing using MLST.Strain typing is a microbial characterizatioz process to distinguish members of the same species at the strain level. Multilocus sequence typing (MLST) refers to the systematic sequencing of six or seven well-conserved, house-keeping genes or loci within the bacterial genome.Allelic variation at each locus is catalogued, and a sequence type is assigned by comparing the set of alleles to other isolate profiles in the database.
-Was also introduced to AMR databases ;
* Greatlife project- Continued to resolve conflicts that arose from The fulltext review
* Attended Greatlife Study Collaborative meeting held at CGHR Kisumu on 30/10/2023

_Plan_
* Continue with resolving conflicts,
* Prepare for the Journal club presentation,

### Latifah Benta
* Attended Greatlife study collaborative meeting and Site visit by the PI, FRank Aarestrup held at CGHR on 30/10/2023
* Worked on resolving the fulltext review conflicts on Covidence
* Attended AMR bioinformatics class on Wednesdays Afternoon. We were taught on MLST (Strain Typing ) and AMR databases.

_Challanges_
- Encountered some difficulties in script-writing,

### Collins Kigen
_Updates_
* Library prep and sequencing of Enterococcus faecium/faecalis phages
* Participated in submission of Open Innovation Program grant proposal aimed at preclinical and clinical studies to validate the Seegene’s multiplex 4-target PCR panel for (i) S. aureus identification, detection of (ii) SCCmec/orfX junction, (iii) mecA/mecC gene, (iv) PVL along with an endogenous internal control from skin/nasal swab samples.
* Reformatted and Submitted mcr-10 manuscript as an observation paper
* Meeting with WRAIR IT support team to discuss AMR Genomics IT needs

_Plan_
* PDP2 exercise
* Inventory of supplies for molecular reagents and lab chemical to be displaced
* Develop K. pneumoniae bioinformatics workflow
* Complete drafting KASH abstracts

### Consolidated updates
#### AMR (CK)
* Library prep and sequencing of 17 _E. faecium/faecalis_ phages on Illumina
* QC raw reads from the pahge sequencing; Good quality reads
* Bioinformatics analysis for the Joint EID project, **"Genomic Epidemiology of high-risk pandemic clones of MDR Escherichia coli and Klebsiella pneumoniae in East Africa and Mediterranean regions"**
* Participated in submission of Open Innovation Program grant proposal
* Reformatted and Submitted *mcr-10* manuscript as an observation paper
* Addition of PANISa a tool for screening insertion sequences in whole genomes; Insertion sequences (IS) play a crucial role in the genomic profile of clinical settings, particularly in the context of OXA genes. OXA β-lactamases, such as OXA-58 and OXA-23, are associated with these IS elements, influencing gene expression and antibiotic resistance in bacteria like Acinetobacter baumannii. ISAba1, for instance, is identified as a key player in regulating OXA carbapenemase genes, making them mobile and affecting their expression
* Bioinformatics class on Strain typing by MLST
_Plan_
* PDP2 exercise
* Inventory of supplies for molecular reagents and lab chemical to be displaced
* Develop K. pneumoniae bioinformatics workflow
* Complete drafting KASH abstracts

#### GREATLIFE (LB)
* Attended Greatlife study collaborative meeting and Site visit by the PI, Frank Aarestrup held at CGHR on 30/10/2023
* Greatlife Scoping Review -Worked on resolving the fulltext review conflicts on Covidence
* Attended AMR bioinformatics class on Wednesdays Afternoon.Introduced to MLST (Strain Typing ) and AMR databases and given asingments on the same.

## October 23 2023
Sign in:
1.Vanessa Natasha
2.Collins Kigen
3.James Wachira
4.Brian Kimutai
5.Latifah Benta
6.Joseph Mugah

### Vanessa Natasha
_Updates_
* P.aeruginosa phages initial analysis( Assembly & annotation) and visulization on R
* Participated in report writing for the phage analysis performed.
* Completed visulization of Strain Types in the E.coli data
* Partcipated in the first EID meeting between Kenya,Uganda and Jordan
* Initial KASH abstract writing

_Challenges_
* Debugging R script for E.coli visualization

_Plan_
* Complete E.coli Visualization.
* Complete the A.baumannii abstract for KASH.
* Screen for insertion sequences on the A.baumannii data.

### Collins Kigen
_Updates_
* Organized a meeting with USAMRD bioinformaticians from Jordan, Kenya and Uganda. The purpose was to plan a collaborative bioinformatics analysis for a joint project on Genomic epidemiology of pandemic MDR E. coli and K. pneumonia strains. We succesfully defined a strategic path and we now have a clear way forward. We agreed on weekly meetups to touch base and ensure progress.
* Attended the 2023 Wastewater Biosurveillance Workshop. The goal of the workshop was to bring together members of the Wastewater Surveillance community, from government, industry and academia, to build community, share ideas, methods & technologies, foster discussions and collaborations, and define what is next for wastewater biosurveillance. My reflection on the workshop is: It was a high-level meeting and lacking the domain expertise my contributions were limited. Nevertheless, It was intruiging to learn the utility of wastewater as an epidemioloigcal tool for detecting and monitoring disease outbreaks such as SARS-COV2, mpox and even HIV.
* Participated in drafting a summary report for WGS of P. aeruginosa phages as part of MIDRIP project.

_Plan_
* Enterococcus phage sequencing
* Analysis of E. coli WGS for joint EID project

### James Wachira
_Updates_
* Performed a quick demo on how to download sequence data from the SRA database and Genome assembly during the Bioinformatics training
* Attended the 1st EID meeting between Kenya, Uganda and Jordan bioinformatic teams
* Made a presentation during the Monday Journal Club meeting held on 16th October 2023
* Took part in drafting a report on some of the MIDRIP phage bioinformatic analysis done so far

_Plan_
* Take part in Soro's Library prep and sequencing of Enterococcus phages
* Streamline One Codex Platform results and CARD output for comparison analysis

### Brian Kimutai

*Updates*
1. GreatLife Project - Held multiple meetings to resolve Conflicts that arose from the Full-text Review;
2. Bioinformatics Training- Attended the weekly Bioinformatics Training, Was introduced to Genome Assembly Using Flye, visualization and Quality control tools ;Bandage and Fastqc respectively.Flye is a Bioinformatics tool used to assemble long reads data from sequencing platforms such as Oxford Nanopore sequencers and Pacbio. Bandage is a tool used for visualization while Fastqc is one for quality checks for raw sequence data.

_Challenges_

- Some articles lack The full text while others require Payment in order to acccess the full text. This really slowed down the Full text review.

*Plan*
- Finish up on resolving Conflicts,
- Attend Bioinformatics training,

### Joseph Mugah

*Updates*
1. Finished full text review of articles for GreatLife project
2. Attended bioiniformatics classes
3. Attended meetings to solve conflicts that arose from full text review
4. Did routine laboratory work

*Challanges*

- Several articles either lacked full text or couldnt be accessed

*Plan*

- Finish resolving conflicts and start extraction process
- Continue with bioninformatics classes

### Latifah Benta
1. Bioinformatics Training- Was introduced to Genome Assembly in the AMR Bioinformatics class. We practiced using tools like Flye, Bandage and FastQC on sequences that we downloaded from NCBI, SRA databases
2. Greatlife project- Held meetings to resolve conflicts that arose from The fulltext review

*Challenges*
1. Most of the articles are not available or are to pay for
2. Most sequences available were too large for our machines

*Plan*
1. Continue with ongoing meetings to finnish resolving conflicts
2. Find a way to manipulate large sequence files in order to assemble and visualize them.

### Consolidated Updates
_AMR_ (VN)
_Updates_
* Intial analysis of the P.aeruginosa phages analysis (MIDRP). Performed assembly and annotation
* Report writing for the P.aeruginosa phage analysis conducted
* Attended and participated in the first EID meeting for bioinformatics analysis of MDR _E.coli_ and _K.pneumoniae_ The purpose was to define the scope of the genomics analysis that would be conducted and to define a strategic plan on how to go about it.

_Plan_
* Library prep for the Eneterococcus phage sequencing on the Illumina platform
* Consolidate a pipeline for E.coli genomics that will be used on the EID analysis of the E.coli sequences

GREATLIFE (LB)
1. Bioinformatics Training- Was introduced to Genome Assembly in the AMR Bioinformatics class. We practiced using tools like Flye, Bandage and FastQC on sequences that we downloaded from NCBI, SRA databases
2. Greatlife project- Held meetings to resolve conflicts that arose from The fulltext review
3. Did routine laboratory work

*Plan*
- Finish up on resolving Conflicts,
- Attend Bioinformatics training,

## October 6th 2023
Sign in:
1. Collins Kigen
2. James Wachira
3. Vanessa Natasha

### Collins Kigen
_Updates_
* WGS of P. aeruginosa phages MIDRIP batch 1 & 2
* Visualization of E. coli genomics analysis results

_Challenges_

_Plan_
* Bioinformatics analysis of P. aeruginosa phages
* Library prep and seq of Enterococcus phages on Illumina

### James Wachira
_Updates_
* Oxford Nanopore sequencing of MIDRIP P.aeruginosa phages
* Performed a multiplex PCR detection of 6 AmpC genes in carbapenem resistant bacteria
* Integrated a new qc tool, INHERIT, useful for distinguishing phage from bacteria contigs with 99% accuracy
* RGI software debugging completed

_Challenges_
* Low number of available pores on flow cell used for MIDRIP batch2 phages

_Plan_
* Bioinformatic analysis of both MIDRIP and A.baumanii phages sequencing data
* Another PCR run for detection of carbapenem genes
* Work on a script for reproducing AmpliSeq results utilizing the CARD database for comparison with OneCodex platform

### Vanessa Natasha
_Updates_
* Library prep for _P.aeruginosa_ phages and subsequent sequencing of the phages
* Gel run of C.difficle ribotypes
* Screening for strain types for the _A.baumannii_ MRSN sequences.
* Visualization of _E.coli_ Strain types (barplot for no of isolates vs st types, and pie chart of abundant strain types)
* Finding a command line tool to screen mobile elements .

_Challenges_
* There was low number of pores available for sequencing of _P.aeruginosa_ phages
* Finding a tool that I can use to screen Mobile elements on the _A.baumannii_ sequences (command line tool)

_Plan_
* Continue with the visualization of E.coli analysis
* P.aeruginosa phages analysis
* Complete the MST script
* Library prep Illumina sequencing for Eneterococcus phages

### Consolidated updates
#### AMR (JW)
##### Updates
1. Library prep and sequencing of P.aeruginosa phages MIDRIP done in 2 batches
2. Multiplex PCR for detecttion of AmpC genes in carbapenem-resistant isolates
3. Integration of an in-house qc tool for differentiating bacteria from phage sequences after performing genome assembly
4. Visualization of E.coli genome analysis is still ongoing
5. Bioinformatics training sessions on Bacterial Genomics and Preprocessing of raw data

##### Plan for the next 2 weeks
1. Library prep and sequencing of Enterococcus phages on Illumina platform
2. Bioinformatic analysis for the P.aeruginosa phages
3. Complete analysis of E coli genomes and draft a report

## September 23rd 2023
Sign in:
1. Collins Kigen
2. James Wachira
3. Vanessa Natasha
4. Joseph Mugah
5. Latifah Benta
6. Brian Kimutai

### Collins Kigen
_Updates_
* WGS of A. baumannii phages on Nanopore
* E.coli genomics analysis: Serotyping, plasmid replicons
* Analysis of MRSA isolates (Justin): AMR genes, Pangenome analysis
* Assisted Polly to generate visualization graphs
* Developed costing with EO for WGS on Nanopore and Illumina sequencing as a service
* Chaired Journal club presentantion by Oumarou Soro "**Characterization of a Novel Bacteriophage Henu2 and Evaluation of the Synergistic Antibacterial Activity of Phage-Antibiotics**"

_Challenges_
* Delay in procurement of nanopore reagents
* Synology server is down
* Missed Kenya/Tanzania/Georgia manuscript meeting due to prolonged lib prep seqeuncing experiment

_Plan_
* Sequence MIDRIP psedomonas phages on nanopore
* Seqeunce Enterococcus phages on Illumina (Soro)
* Guide Felix on bioinformatics analysis of A. baumannii phages
* Draft A. baumanii Genome announcement manuscript
* Reformat mcr10 manuscrit paper from research article to observation paper
* Attend F&S meeting about Bioanalyzer

### James Wachira
_Updates_
* Performed whole genome sequencing of A.baumanii phages on Nanopore alongside Collins, Vanessa, Brian, Benta, Roselyn
* Learnt how to perform flow cell cleaning and storage after use
* Performed 16S PCR on C.difficile bacteria isolates as well as A.baumanii phages followed by gel electrophoresis
* Alongside Collins,we set up nvidia graphics card drivers on 2 machines to facilitate gpu-based basecalling which is more than 10fold faster than CPU-based methods
* Downloaded rgi software to download and index CARD database

_Challenges_
* Failure of rgi software to map raw reads to AMR genes in the CARD database. There is a technical fault in an alignment step within the rgi pipeline and there are no fixes to this problem postedon the rgi community

_Plan_
* Fall back to an earlier version or upgrade rgi and see whether the problem is resolved
* Perform Phage Genomics analysis from sequencing data

### Vanessa Natasha
_Updates_
* 16s PCR run on _C.difficile_ DNA samples followed by subsequent gel electrophoresis for the samples
* Worked with the genomics team (Collins, James, Benta, Brian and Roselyn) on library prep for A.baumannii phages for the Nanopore platform.
* Learned how to set up a Nanopore sequencing run,how to do a flow cell wash and checking the remaining number of pores available for sequencing
* Screened for plasmids on the A.baumannii MRSN sequences
* Worked on an R-script for organising data from Abritamr
* Compilation of quantification results for the phages that have been extracted so far(_A.baumannii_ and _P.aeruginosa_)

_Challenges_
* Debugging the R-script for Minimum Spanning tree (MST) (figure out how to calculate pairwise similarities on R)

_Plan_
* Continue on E.coli analysis
* Continue on A.baumannii MRSN analysis
* Debugging the R-script for Minimum Spanning tree(MST)

### Joseph Mugah

_Updates_

+ Screened 74 articles for the Greatlife Project.
+ Prepared BHI media
+ In collaboration with other staff, received and processed samples for TB diagnosis
+ Attended a refresher course on ISO 15189-2022

_Plan_

+ Finish reviewing of articles
+ Continue with Bioinformatics classes

## Latifah Benta
_Updates_
+ Screened for articles on the Covidence app for the Greatlife project
+ Performed PCR on _A.baumanii_ phages to check for contamination by bacteria.
+ Confirmed _C.difficile_ samples by running PCR to check for the 16s region before ribotyping
+ Learnt how to quantify DNA using the Qubit fluorometer
+ Did Whole Genome sequencing on Oxford nanopore platform

_Challenges_
+ Smears in the gel when we did the _C.difficile_ PCR
+ Non-specific amplification during the _A.baumanii_ phages PCR run

_Plans_
+ Finish on the Greatlife fulltext review
+ Work on my presentation for the journal club

## Brian Kimutai
_Updates_
1. Did Full text review of Articles For the GREATLIFE project's Scopping review
2. Learned on how to Quantify DNA using the Qubit Fluorometer.The Qubit fluorometer uses fluorescent dyes to determine the concentration of either nucleic acids or proteins in a sample. This method uses a spectrophotometer to measure the natural absorbance of light at 260 nm (for DNA and RNA) or 280 nm (for proteins)
3. Did 16S PCR for A. baumannii phages to confirm absence of bacterial host DNA
4. Did Whole Genome sequencing using Oxford nanopore sequencing Technology{ONT};

_Plans_
+ Work on the GREATLIFE Project's fulltext review
+ Attend Bioninformatcics trainning
+

### Consolidated updates
#### AMR (CK)
##### Updates
1. Ampliseq sequencing. Performed pilot experiment
2. Setup in-house bioinformatics analysis for Ampliseq results
3. 16S PCR for A. baumannii phages to confirm absence of bacterial host DNA
4. 16S PCR for C. difficile to assess integrity prior to ribotyping experiment
5. WGS of A. baumannii phages on Nanopore
6. E. coli epidemiology. Performed serotyping
7. Screening for plasmids on A.baumannii MRSN sequences
8. Students support.
a. Justin: Pangenome analysis
b. Polly: Visualization of results on graphpad
c. Roselyne: mecA & mecC screening in EQA MRSA query isolate
d. Soro: Qubit quantification and 16S PCR for Enterococcus phages
e. Winnie: 16S PCR for C. difficle isolates
f. Bioinformatics training session for trainees to complete tasks
##### Plan for next 2 weeks
1. Nanopore sequencing of MIDRIP psedomonas phages
2. Illumina sequencing of Enterococcus phages
3. Reformat mcr-10 manuscript as observation paper
4. Complete analysis of E coli genomes and draft a report
5. Test new work model to improve productivity
* 1st 2 weeks of the month for lab work:
* Routine screening of mecA and mecC genes in MRSA
* AmpliSeq
* Nanopore
* Illumina
* 3rd week of month for Bioinformatics analysis
* 4th week of month for writing reports/manuscripts/meetings

#### Greatlife Project (LB)
+ Fulltext review on Covidence. We currently have 388 articles left
+ Whole Genome Sequencing on _A.baumanii_ phages on Oxford nanopore
+ Detection of bacterial host contaminant in _A.baumanii_ phage DNA using PCR
+ Received and processed samples for TB
+ Prepared Brain Heart Infusion media

## Plan for the next 2 weeks
+ Finish the fulltext review for the greatlife project.
+ Attend and work on the Bioinformatics training.
+ Conduct routine laboratory work.

## September 11th 2023
Sign in:
1. Collins Kigen
2. Vanessa Natasha
3. James Wachira
4. Brian Kimutai
5. Joseph Mugah
6. Latifah Benta

### Collins Kigen
_Updates_
* Guided attachees project on "Validation of MRSA by PCR" (Roselyn and Vanessa)
* Chaired Journal club presentantion by Cecilia “My Science Journey: Scholarships and studying abroad”
* Participated in drafting a report on AmpliSeq sequencing "AmpliSeq AMR panel: Evaluation of its utility in detection of antimicrobial resistance genes in clinical priority pathogens "
* Drafted genomics monthly report
* Trained AMR team on molecular SOPs
* Particpated in updating, reviewing and filing of Lab SDSs
* Attended webinar on "Vaccines: A Critical Tool in the Fight Against Antimicrobial Resistance webinar" (Aug 29th)
* Attended AMR/MHK lab floor plan meeting (Aug 29th)

_Challenges_
* Unable to access the synology storage server as it is currenly offline; retrival of WGS for analysis on hold

_Plan_
* Library prep and illumina sequencing of 16 A. baumannii phages
* Reformat mcr-10 manuscript as observation paper and resubmit to ASM journal
* Review and return Polly's manuscript
* Meeting with Justin on manuscript progress
* Attend meeting with F&S representantive regarding Bioptic bioanalyzer

### Vanessa Natasha
_Updates_
- Training of interns and students on Bash scripting (loops, grep, sed )
- One codex analysis for ampliseq tarageted sequencing
- Compilation of OneCodex analsysis output and AST reports
- Visualization script for onecodex output ( a heatmap for the AMR gene carriage for samples )
- Partcipated in drafting the Ampliseq report:- "AmpliSeq AMR panel: Evaluation of its utility in detection of antimicrobial resistance genes in clinical priority pathogens"
- PCR run for 16s bacteriophage samples to detect host DNA ( Soro's project)
- Reviewing all SDS for the different labs.
- Updating the SDS for all the new reagents in the Master Mix laboratory

_Challenges_
- Accessing some SDS for some master mix reagents ( some of these reagents have no sds from the manufacturer)

_Plan_
- Work on A.baumannii MRSN analsysis
- MST script for E.coli
- Library Prep for 16 A.baumannii phages

### James Wachira
_Updates_
- Presented a brief on Monday 4th Sept 2023 on protein structure modelling workflow for the phoQ protein, a component of the mcr10 manuscript
- AMR gene detection on the OneCodex platform for the AmpliSeq pilot study raw read data from Illumina
- Did a comparative analysis of Whole Genome Sequencing raw reads and AmpliSeq raw reads for the control samples using the OneCodex platform
- Participated in drafting of the AmpliSeq report on the 32 MDR/MRSA samples while evaluating AmpliSeq's advantages/limitations
- Worked on an ad-hoc python script for plotting the frequency of AMR genes across the bacterial isolates in the study while highlighting the major associated antibiotic classes
- Participated in reviewing,modifying and updating Material Safety Data Sheets for new and existing chemicals in the lab
- Wilfred is currently working on fixing the synology storage server which has the only copy of the Enterobacter sequences I need to start bioinformatic analysis on
- Was able to do a training on the command line application of the collaborative github platform on Wednesday 6th Sept 2023

_Challenges_
- Tedious nature of doing manual frequency counts while using Excel sheets. A need to learn more automation techniques
- Unpublished SDS documents for certain chemicals especially ones manufactured locally

_Plans_
- Reviewing the use of CARD and RGI as a tool for targeted sequencing bioinformatic analysis as we try to implement a custom One Codex bioinformatics analysis workflow
- Reviewing literature in the search of a relevant antibiotic that I can focus on even as we continue to do analysis on AMR genes and resistance mechanisms against susceptible antibiotics

### Brian Kimutai
_Updates_
- Attended Bioinfromatics training on Bash scripting(loops,grep and sed) and On working with Git on the Command line
- Continued with the GREATLIFE Projects' Full text review, 57 Articles are ready for Extraction 428 Articles are still not yet fully reviewed
- Participated in running PCR for bacteriophage samples under the guidance of Vanessa
- Participated in reviewing SOPs
- Held a briefing meeting for the GREATLIFE Project
- Attended Journal club presentantion by Cecilia “My Science Journey: Scholarships and studying abroad”

_Plan_
- To continue with the Full Text review of articles for the GREATLIFE Project
- Attend meeting with Eddy to Report on Progress with Full text Review
- Attend Bioinformatics Training

### Joseph Mugah
_Updates_

- Attended the monthly laboratory meeting
- Reviewed 113 aerticles on covidence for GREATLIFE Project
- In collaboration with other laboratory staff, we processed various TB samples and did other routine laboratory work
- Attended bioninformatics training on Shell Scripting and Git on the Commandline
- Attended a brief session with my colleagues where we gave a status report on the progress of full text review of articles
- Attended Journal club presentantion by Cecilia “My Science Journey: Scholarships and studying abroad”

_Plan_
- To complete full text review of articles for GREATLIFE Project and embark on extraction
- Continue with Bioinformatics class and routine laboratory work
- Attend CMEs sessions scheduled for the week

### Latifah Benta

*Updates*

- Screened 57 articles for the Greatlife project full text review.
- Attended the Bioinformatics training on Wednesday and was introduced to git on the commandline.
- Helped Oumarou Soro run PCR for Bacteriophages under Vanessa Natasha
- Helped with reviewing inventories for Freezer 3 which is in the amplification room
- Helped with organizing SDS binder with Vanessa

*Plan*
- Finnish reviewing articles for the Greatlife project
- Work on the Bioinformatics assignment
- Read on Bash scripting especially on loops

### Consolidated updates
#### AMR (CK)
1. AmpliSeq sequencing and data analysis. AmpliSeq is a new sequencing approach that we are introducing in our lab that involves targeted sequencing of only AMR gene in bacterial isolates unlike WGS that sequences the entire genome. The genomics team have performed the pilot run using 30 samples which were the most recent MDR and/or MRSA isolates as well as 2 control samples whose whole genome has already been sequenced. The aim was to evaluate its utility in detection of AMR for clinical pathogens that we handle. From the analysis we established that AmpliSeq is effective in detection of AMR genes upto 100% compared with WGS. Also the analysis on the One Codex platform that takes in the raw reads from sequencer and returns a summary report with all the AMR genes detected is fast enabling quick turnaround and hence impact positelvy of decision makking in pathogen surveillance
2. 16S PCR for detection of bacterial host dna in phage dna. The genomiocs team guided and assisted Soro in the PCR experiment.

#### GREATLIFE (BK)
1. Full text review - Conducting a Scoping review involves various steps; Defining the Review Objectives and Question(s),Creating the Search Strategy,Conducting the Search, Selecting Studies to be Included ,Performing Data Extraction and Interpreting Findings and finally Presenting the Findings. Full text review is part of the screening process of articles,it involves carefully and critically reading an entire research article before data extraction.Of 519 articles, we have done full text review of 66 articles, 8 have been excluded and 57 are ready for data extraction.
2. Bioinformatics training- We attended the weekly Bioinformatics traning and were introduced to Working with Git on the commandline.
3. 16S PCR for detection of bacterial host dna in phage dna.Under the guidance of Vanessa and James, we took part in running Soro's PCR experiment.