Quality control assessment for microbial genomes: GalaxyTrakr MicroRunQC workflow

This protocol will walk the user through various aspects of the quality assessment of bacterial genome sequences, from setting up a GalaxyTrakr account to the quality control (QC) benchmarks GenomeTrakr uses for its sequencing efforts. For quick access, GenomeTrakr QC benchmarks are included in the table below.

These are also relevant for NARMS and VetLIRN contributors.

*MicroRunQC users should follow QC threshold guidelines established by their respective surveillance coordinating body(s).

A	B	C	D	E	F	G	H	I	J
A	B	C	D	E	F	G	H	I	J
Quality metric	Salmonella	Listeria	E. coli	Shigella	Campylobacter	Vibrio para.	Cronobacter	Enterococcus faecium	Enterococcus faecalis
Average read quality Q score for R1 and R2	>=30	>=30	>=30	>=30	>=30	>=30	>=30	>=30	>=30
Average coverage	>=30X	>=20X	>=40X	>=40X	>=20X	>=40X	>=20X	>=50X	>=40X
De novo assembly: Seq. length (Mbp)	~4.3-5.2	~2.7-3.2	~4.5-5.9	~4.0-5.0	~1.5-1.9	~4.8-5.5	~4-5	~2.5-3.5	~2.5-3.25
De novo assembly: no. contigs	<=300	<=300	<=400	<=550	<=300	<=300	<=500	<=350	<=200

Create a GalaxyTrakr account here: https://account.galaxytrakr.org/Account/Register

Log into your GalaxyTrakr account: https://galaxytrakr.org

Create a new history.

We recommend creating a new history for each new MiSeq Run and including the flow-cell ID and date in the history name.

Save your MicroRunQC output here and any other relevant analyses, like serotyping, or AMR detection.

After all the analysis output from this run is saved to your internal data network or computer, older histories should be purged/deleted so as not to occupy the limited storage space in your account. In some cases it may be useful to save, for a limited time, multiple histories or to run analyses concurrently in multiple histories. In these cases you need to pay attention to your % usage bar (shows % used of allocated storage space) in the upper right corner of the GalaxyTrakr page. If you need additional space you can contact galaxytrakrsupport@fda.hhs.gov and request additional storage.

Click on the + icon in the upper right History panel

Name your new History by clicking on the “Unnamed history" text, type in desired name, and hit Enter. We recommend including the run cell ID and the date the run was started.

This section will describe the process for uploading raw fastq files into your active History panel. After the files have been uploaded they will stay in your account until they are deleted.

Click on the Upload Data icon on the top of the left web page to start an upload process.

Select " Type (set all):auto-detect. " Click " Choose local files " button and navigate to the desired fastq files, then click " Start " to upload files. These files should be paired (two per sample/isolate).

As the file uploads complete, each row will turn green. Samples in yellow are still in process.

You have just upload a set of forward and reverse reads. For further analysis, these files need to be paired properly so the platform knows which R1 and R2 files go together. GalaxyTrakr does this by creating a List of Dataset Pairs.

Within your newly created History panel, click the check box, then select all the files you just uploaded by clicking "All" or by individually selecting the ones you want to pair.

Click "For all selected" and choose "Build List of Dataset Pairs"

A new window will open to help you pair the fastq files properly. Note how your paired reads are named.

First, click on the drop down arrow and choose “_R1,” if that is the naming convention your files follow. This automatically populates the corresponding “_R2” in the next box.​

Click Auto-pair.

Paired reads will pair in the middle column and turn green.

Unselect " Hide original elements," which is the default setting and undesired here.

<img src="https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.5jyl8mj16g2w/pairing_data_hide_original_elements.png" alt="Clear the checkmark by "Hide original elements?" seen here by clicking on it." loading="lazy" title="Clear the checkmark by "Hide original elements?" seen here by clicking on it."/>

Name your dataset: Example, "pairedSet--"

Click Create list.

This paired dataset will now be available for analysis in your history panel. You can run multiple analyses on the same dataset in a history rather than upload the same sequence data to a new history to perform additional analyses. This will help you use your allocated storage space efficiently.

Add the MicroRunQC workflow to your own "Workflows" panel. You only have to do this step once for each new workflow you need.

Navigate to the “ Shared Data " drop down menu, choose " Workflows," and search for "MicroRunQC_v1.2."

From the dropdown menu by the MicroRunQC title, select " Import. "

To see the new imported workflow, click the “ Workflow ” tab on the top panel.

Click the box under " Bookmarked " to make it available in the left panel under " Workflows " when MicroRunQC is searched for.

From the Workflow menu on the left panel, select MicroRunQC_v1.2

Select paired list dataset you created earlier.

Click Run Workflow . This can take some time depending on the number of samples you are analyzing. If you choose to you can log out of GalaxyTrakr and log back in at a later time to see if the job is completed.

Upon completion of the pipeline all tiles in the History pane will be green.

In the “ Filter on Data ## ” tile, click on the “Eye” icon to view the output table in the GalaxyTrakr window.

Download and interpret the results:

Click Filter on data ## and then the floppy disc icon. The tabular file can be opened in a text reader or converted to a format (.txt) that can be opened in Excel.

The MicroRunQC output file includes the following columns:

Parameter | A | B | C | | --- | --- | --- | | Parameter | Input | Description | | Contigs | Assembly | Number of contigs in the de-novo SKESA assembly. Contigs smaller than 200 base-pairs (bp) are not counted.  | | Length | Assembly | Total length of all contigs > 200bp. This should approximate the size of the genome for the target organism. | | EstCov | Assembly | Mean coverage for contigs in the SKESA assembly. | | N50 | Assembly | Sequence length of the shortest contig at 50% of the total genome length | | MedianInsert | Read | Distance between forward and reverse reads. Calculated by mapping reads to SKESA assembly using bwa. | | MeanLength_R1 | Read | Mean length of forward read | | MeanLength_R2 | Read | Mean length of reverse read | | MeanQ_R1 | Read | Mean Q-score of forward read | | MeanQ_R2 | Read | Mean Q-score of reverse read | | Scheme | Assembly | PubMLST scheme name (output from mlst application that scans contig files against traditional PubMLST typing schemes. | | ST | Assembly | Sequence Type  | | MLST extra | Assembly | e.g. Listeria clonal complex info | | Loci | Assembly | gene (allele number) – for example aroC(118) |

MicroRunQC output table headers. This table lists the summary metrics for sequence quality, number of contigs, and estimated genome size, along with other common metrics for reads (Median Insert Size and Mean Length) and assemblies (N50). Additionally, if the Multi-Locus Sequence Type (MLST) for the isolate is available from pubmlst, the workflow also reports Sequence Type (ST) and the associated alleles.*MLST extra: Additional data fields reported when available in Sequence Type definition files (not available for all species)1. clonal_complex – sequences grouped by similarity to central allelic profile (e.g., Campylobacter ST-21 complex)2. CC – clonal_complex – Abbreviation used for organism like Listeria, ST profiles are maintained by different groups3. Lineage – Listeria monocytogenes lineage (I,II,III, and IV), Listeria species also reported here (e.g.L.innocua)4. species – e.g., Vibrio alginolyticus Input Description Contigs Assembly Number of contigs in the de-novo SKESA assembly. Contigs smaller than 200 base-pairs (bp) are not counted. Length Assembly Total length of all contigs > 200bp. This should approximate the size of the genome for the target organism. EstCov Assembly Mean coverage for contigs in the SKESA assembly. N50 Assembly Sequence length of the shortest contig at 50% of the total genome length MedianInsert Read Distance between forward and reverse reads. Calculated by mapping reads to SKESA assembly using bwa. MeanLength_R1 Read Mean length of forward read MeanLength_R2 Read Mean length of reverse read MeanQ_R1 Read Mean Q-score of forward read MeanQ_R2 Read Mean Q-score of reverse read Scheme Assembly PubMLST (pubmlst.org) database scheme (e.g. senterica for Salmonella enterica) ST Assembly Sequence Type Loci Assembly gene (allele number) – for example aroC(118)

**This output should be saved either to your LIMS or to a spreadsheet linked to the sequencing run and samples.

Example output for 1 Salmonella and 5 Listeria isolates.

A	B
Srain ID	Lab Confirmation
FDA1216271-C001-001	Listeria mono
FDA817806-S073-001	Listeria mono
FDA746634	Listeria mono
FDA1213377-C001-002	Listeria grayi
FDA933376-S060-005	Listeria innocua
FDA1213835-C001-001	Salmonella

Lab confirmed IDs for 6 isolates

A	B	C	D	E	F	G	H	I	J	K	L	M	N	O	P	Q	R	S	T
File	Contigs	Length	EstCov	N50	Median Insert	Mean Length_R1	Mean Length_R2	Mean Q_R1	Mean Q_R2	Scheme	ST	MLST extra
FDA1216271-C001-001	16	2911949	36.7	476210	321	148.4	148.4	36.4	34.6	listeria_2	5	CC=CC5,Lineage=I	abcZ(2)	bglA(1)	cat(11)	dapE(3)	dat(3)	ldh(1)	lhkA(7)
FDA817806-S073-001	20	3068354	179.6	525438	329	234.7	235.2	36.7	31.9	listeria_2	321	CC=CC321,Lineage=II	abcZ(5)	bglA(6)	cat(8)	dapE(62)	dat(6)	ldh(7)	lhkA(34)
FDA746634	30	3052888	41.4	293947	320	148.4	148.4	36.5	36	listeria_2	-		abcZ(2)	bglA(1)	cat(11)	dapE(3)	dat(3)	ldh(1)	lhkA(~7)
FDA1213377-C001-002	20	2672180	155.1	473181	270	147.3	147.3	37.2	36.1	-	-
FDA933376-S060-005	9	2881869	213	1498790	303	232.1	232.2	37	36.2	listeria_2	1489	CC=CC1489,Lineage=L. innocua	abcZ(250)	bglA(21)	cat(83)	dapE(298)	dat(20)	ldh(458)	lhkA(216)
FDA1213835-C001-001	37	4832365	34.4	294936	354	149	149	36.6	35.7	senterica_achtman_2	214		aroC(14)	dnaN(72)	hemD(21)	hisD(12)	purE(6)	sucA(19)	thrA(15)

MicroRunQC example report showing mlst ST results for different Listeria species.

The mlst Listeria database includes multiple species, including Listeria monocytogenes and L. innocua . When available, the Listeria clonal complex (CC) or L. monocytogenes lineage is listed alongside the ST.

For quality control threshold guidelines for the GenomeTrakr surveillance network, These are also relevant for NARMS and VetLIRN contributors.

*MicroRunQC users should follow QC threshold guidelines established by their respective surveillance coordinating body(s).