Output files

Output files for sequencing

The following table is a list of Bases2FastqDx outputs files for sequencing:

File	Directory	Description
Bases2Fastq.log	info	Log file that records software events
IndexAssignment.csv	Root	Yield, number, and rate of polonies that are assigned for each sample and index combination
Metrics.csv	Root	Mismatch rates, percent assigned, and per sample yield for each lane
MultiQC_report.html	Root	MultiQC report on the performance and quality of the sequencing samples
{ProjectName}_QC.html	Samples/{ProjectName}	Interactive HTML QC report on the performance and quality of the samples aggregated by project
{ProjectName}_index_assignment.csv	Samples/{ProjectName}	Yield, number, and rate of polonies that are assigned for each sample and index combination in a project
{ProjectName}_metrics.csv	Samples/{ProjectName}	Mismatch rates, percent assigned, and per sample yield for each lane in a project
{ProjectName}_RunStats.json	Samples/{ProjectName}	Information on the performance of samples in a project
{RunName}_QC.html	Root	Interactive HTML QC report on run performance and quality for all samples and projects
RunManifest.csv	Root	Run manifest for the Bases2FastqDx execution
RunManifest.json	Root	Machine-readable copy of the run manifest as a JSON file
RunManifestErrors.json	info	Record of errors in the run manifest
RunParameters.json	Root	Copy of the original run parameters file
{SampleName}_{Read}.fastq.gz	Samples/{SampleName} or Samples/{ProjectName}/{SampleName}	The primary output of Bases2FastqDx
{SampleName}_stats.json	Samples/{SampleName}	Information on the performance of each sample in the run
UnassignedSequences.csv	Root	The most frequent unassigned index sequences with approximate counts[^1]

[^1] ^{Counts indicate how many times an incorrect index sequence appears.}

FASTQ files

A FASTQ file records all genomic data and corresponding Q scores for a sample or cell. FASTQ files are GZIP-compressed text files that Bases2FastqDx names {SampleName}_{Read}.fastq.gz for sequencing, and {Well}_{Batch}_R1.fastq.gz and {TargetSite}_{Well}_{Batch}_R1.fastq.gz for untargeted and targeted cytoprofiling runs respectively.

Each entry in a FASTQ file corresponds to one read and includes the following four lines:

• A sequence identifier that includes run and polony information
• Base calls that are assembled into a sequence comprised of A, C, G, T, and N
• A plus sign (+) that separates the sequence from the Q scores
• A Q score for each base in the sequence

Note:

If you use a sequencing run manifest with no samples or associated index sequences, then Bases2FastqDx assigns all reads to DefaultSample. The software only produces the FASTQ files DefaultSample_R1.fastq.gz and DefaultSample_R2.fastq.gz.

Sequence identifiers for sequencing

The sequencing FASTQ sequence identifier includes the components in the following table and is formatted in one line:

@<instrument>_<model>:<run name>:<flow cell ID>:<lane>:<tile>:<x-pos>:<y-pos>:UMI <read>:N:0:<index sequence>

Component	Allowed Values	Description
@	@	Start to the sequence identifier line
<instrument>_<model>	Upper and lowercase letters, integers 0–9, dashes (-), and underscores (_)	Instrument name, suffixed with an underscore and the instrument model (for example, _AVITI) when RunParameters.json reports the model.
<run name>	Upper and lowercase letters, integers 0–9, hyphens (-), and underscores (_)	Run name that is defined during the run setup
<flow cell ID>	Upper and lowercase letters and integers 0–9	Flow Cell ID from the barcode scan. If the barcode scan fails during the run and no barcode is present, then the Run ID replaces the Flow Cell ID.
<lane>	1 or 2	Lane number
<tile>	An integer	Tile number
<x_pos>	A zero-padded integer	X-coordinate of the polony
<y_pos>	A zero-padded integer	Y-coordinate of the polony
<UMI>	A, C, G, T, and N	UMI sequence with a plus sign that separates the Read 1 and Read 2 sequences, if applicable
<read>	1 or 2	Read number
<is filtered>	N	A legacy filtering value of N that exists only for backward compatibility and does not change
<control number>	0	A legacy control number of 0 that exists only for backward compatibility and does not change
<index sequence>	Varies	A value that is based on the indexing strategy that is indicated in the run manifest: No indexing: The sample number Single indexing: The observed index sequence Dual indexing: The observed Index 1 sequence, a plus sign, and the observed Index 2 sequence

Quality scores

A Q score is based on the Phred scale and indicates the confidence of a base call. A Phred quality score (Q) is logarithmically related to error rate (E): Q = -10log E.

In a FASTQ file, an ASCII code represents the Q score. Bases2FastqDx encodes quality scores with a +33 offset (Phred33).

Q Score	ASCII Code	Character		Q Score	ASCII Code	Character		Q Score	ASCII Code	Character
0	33	!		19	52	4		38	71	G
1	34	"		20	53	5		39	72	H
2	35	#		21	54	6		40	73	I
3	36	$		22	55	7		41	74	J
4	37	%		23	56	8		42	75	K
5	38	&		24	57	9		43	76	L
6	39	'		25	58	:		44	77	M
7	40	(		26	59	;		45	78	N
8	41	)		27	60	<		46	79	O
9	42	*		28	61	=		47	80	P
10	43	+		29	62	>		48	81	Q
11	44	,		30	63	?		49	82	R
12	45	-		31	64	@		50	83	S
13	46	.		32	65	A		51	84	T
14	47	/		33	66	B		52	85	U
15	48	0		34	67	C		53	86	V
16	49	1		35	68	D		54	87	W
17	50	2		36	69	E		55	88	X
18	51	3		37	70	F		56	89	Y

HTML QC reports

HTML QC reports are organized into tabs that display histograms and other charts. The charts visualize index assignment and other quality metrics. If the run manifest includes more than 120 samples, then the report does not display per sample charts.

Caution:

Review QC metrics to confirm that run quality meets expected thresholds. If QC metrics fall outside acceptable ranges, do not proceed with downstream diagnostic analysis.

Bases2FastqDx names the run-level QC report {RunName}_QC.html and the project-level QC reports {ProjectName}_QC.html.

HTML QC reports for individually addressable lanes

To generate HTML QC reports for each lane, create projects for each lane in your run manifest. For an example, see the Run Manifest Documentation.

Missing HTML QC report

If an HTML QC report does not generate on a system that is configured to run the static binary, then complete the following troubleshooting steps:

• Make sure that compatible versions of Python and the necessary packages are installed.
• To identify the cause, review the error in info/QCReportErrors.txt. Then, use this information to generate the HTML QC report.

MultiQC reports

MultiQC reports, which are generated by Seqera and designed by Element Biosciences, are available for use with Bases2FastqDx. MultiQC reports analyze results and statistics from bioinformatics tool outputs, such as log files and console outputs. These reports help summarize experiments that contain multiple samples and multiple analysis steps and are designed to be placed at the end of pipelines or to be run manually when you finish running your tools. Furthermore, MultiQC reports contain the parsed data in a structured format, ready for any further downstream analysis. For more information, see MultiQC Documentation.

Metrics files

Bases2FastqDx reports metrics in different files and formats to support different use cases:

• Metrics.csv offers a high-level overview of yield and assignment metrics, per lane and overall.
• IndexAssignment.csv summarizes index assignment rates per sample-index pair, per lane, and overall. The project-level index assignment CSV files provide metrics at the level of specific projects.
• The JSON metrics files provide aggregate metrics at the run, project, and sample levels with more details than the summary files. The sample-level files also provide metrics at the level of specific occurrences.

Run metrics for sequencing

The run metrics file RunStats.json reports the following sequencing run performance metrics in JSON format. These metrics are specific to the Bases2FastqDx execution.

Metric	Value
AnalysisID	The unique identifier that Bases2FastqDx generates for the analysis
AnalysisVersion	The current version of Bases2FastqDx
AssignedYield	The run yield that is based on assigned reads in gigabases
FileVersion	The current version of the file format
FlowCellID	A flow cell identifier that is sourced from RunParameters.json. If blank, then the letter R followed by the RunID value is used.
I1IsReverseComplement	The observed orientation of the Index 1 sequences relative to the orientation recorded in the run manifest
I2IsReverseComplement	The observed orientation of the Index 2 sequences relative to the orientation recorded in the run manifest
Lanes	A detailed list of per lane metrics
MeanReadLength	The average read length after adapter trimming
NumPolonies	The total number of polonies that are calculated for the run
NumPoloniesBeforeTrimming	The total number of polonies that are calculated for the run before adapter trimming
PercentAssignedReads	The percentage of reads that are assigned to a sample
PercentBelowFilterThreshold	The percentage of all polonies that are below the quality filtering threshold for each cycle
PercentMismatch	The percentage of polonies that are assigned to a sample with a mismatch
PercentMismatchI1	The percentage of polonies that are assigned to Index 1 sequences with a mismatch
PercentMismatchI2	The percentage of polonies that are assigned to Index 2 sequences with a mismatch
PercentQ30	The percentage of ≥ Q30 Q scores for the run. This includes assigned and unassigned reads and excludes filtered reads and no calls.
PercentQ40	The percentage of ≥ Q40 Q scores for the run. This includes assigned and unassigned reads and excludes filtered reads and no calls.
PercentReadsTrimmed	The percentage of reads that Bases2FastqDx trimmed
PercentUnexpectedIndexPairs	The percentage of all polonies with Index 1 and Index 2 reads that matched different samples[^1]
PerReadMeanQualityScoreHistogram	The distribution of per-read average quality scores
QualityScore10thPercentile	The 10th percentile of quality scores
QualityScore25thPercentile	The 25th percentile of quality scores
QualityScore50thPercentile	The 50th percentile of quality scores
QualityScore75thPercentile	The 75th percentile of quality scores
QualityScore90thPercentile	The 90th percentile of quality scores
QualityScoreHistogram	A per-base call Q score distribution with integer resolution
QualityScoreMean	The mean Q score of base calls for the run and excludes filtered reads and no calls
RemovedAdapterLengthHistogram	A histogram that shows the number of bases trimmed from an adapter at a given position
RunName	A text-based run identifier that is sourced from RunParameters.json
RunID	A universally unique identifier (UUID) that is assigned to the run and sourced from RunParameters.json
Samples	A list of libraries that the run sequenced
SampleStats	The per-sample metrics that are listed in the sample metrics files for the run
TotalYield	The total yield of all reads in gigabases
UnassignedSequences	A list of unassigned index sequences with a count for each unassigned sequence

[^1] ^{For demultiplexing to be successful, both index reads must match the same sample.}

Project metrics for sequencing

When a run manifest groups samples by project, Bases2FastqDx creates JSON project metrics files. Bases2FastqDx names the files {ProjectName}_RunStats.json. The files report the following performance metrics for the samples in the project:

Metric	Value
AnalysisID	The unique identifier that Bases2FastqDx generates for the analysis
AnalysisVersion	The current version of Bases2FastqDx
BaseComposition	Counts for each A, C, G, T, and N base
FileVersion	The current version of the file format
FlowCellID	A flow cell identifier that is sourced from RunParameters.json. If blank, then the letter R followed by the RunID value is used.
I1IsReverseComplement	The observed orientation of the Index 1 sequences relative to the orientation recorded in the run manifest
I2IsReverseComplement	The observed orientation of the Index 2 sequences relative to the orientation recorded in the run manifest
Lanes	A detailed list of per-lane metrics
MeanReadLength	The average read length after adapter trimming
NumPolonies	The total number of polonies that are calculated for the samples in the project
NumPoloniesBeforeTrimming	The total number of polonies that are calculated for the samples in the project before adapter trimming
PercentMismatch	The percentage of polonies that are assigned to samples with a mismatch in the project
PercentMismatchI1	The percentage of polonies that are assigned to Index 1 sequences with a mismatch
PercentMismatchI2	The percentage of polonies that are assigned to Index 2 sequences with a mismatch
PercentQ30	The percentage of ≥ Q30 Q scores for the project. This includes assigned and unassigned reads and excludes filtered reads and no calls.
PercentQ40	The percentage of ≥ Q40 Q scores for the project. This includes assigned and unassigned reads and excludes filtered reads and no calls.
PercentReadsTrimmed	The percentage of reads that Bases2FastqDx trimmed
PerReadGCCountHistogram	A list of counts: the value at index i is the number of reads with i G/C calls
Project	The alphanumeric project identifier
QualityScore10thPercentile	The 10th percentile of quality scores
QualityScore25thPercentile	The 25th percentile of quality scores
QualityScore50thPercentile	The 50th percentile of quality scores
QualityScore75thPercentile	The 75th percentile of quality scores
QualityScore90thPercentile	The 90th percentile of quality scores
QualityScoreMean	The mean Q score of base calls for the samples in the project and excludes filtered reads and no calls
Reads	A detailed list of per-read metrics
RemovedAdapterLengthHistogram	A histogram that shows the number of bases trimmed from an adapter at a given position
RunName	A text-based run identifier that is sourced from RunParameters.json
RunID	A UUID that is assigned to the run and sourced from RunParameters.json
Samples	A list of libraries that are sequenced for the project
SampleStats	The per-sample metrics that are listed in the sample metrics files for the project
SampleID	A globally unique sample identifier
SampleName	The alphanumeric sample identifier
SampleNumber	The numeric sample identifier
Yield	The number of bases in the project in gigabases

Sample metrics for sequencing

Sample metrics files report the following sample-specific performance metrics in JSON format. Bases2FastqDx names the files {SampleName}_stats.json.

Metric	Value
AnalysisVersion	The current version of Bases2FastqDx
BaseComposition	Counts for each A, C, G, T, and N base
ExternalID	An external ID that is specified in the run manifest, if applicable
FileVersion	The current version of the file format
MeanReadLength	The average read length after adapter trimming
NumPolonies	The total number of polonies that are assigned to the sample
NumPoloniesBeforeTrimming	The number of polonies that are assigned to a sample before adapter trimming
Occurrences	Additional information per occurrence of the sample
PercentMismatch	The percentage of polonies that are assigned to a sample with a mismatch
PercentQ30	The percentage of ≥ Q30 Q scores for the sample. This includes assigned reads and excludes filtered reads and no calls.
PercentQ40	The percentage of ≥ Q40 Q scores for the sample. This includes assigned reads and excludes filtered reads and no calls.
PercentReadsTrimmed	The percentage of reads that Bases2FastqDx trimmed
PerReadGCCountHistogram	A list of counts: the value at index i is the number of reads with i G/C calls
QualityScoreMean	The mean Q score of base calls for the sample and excludes filtered reads and no calls
RemovedAdapterLengthHistogram	A histogram that shows the number of bases trimmed from an adapter at a given position
RunName	A text-based run identifier that is sourced from RunParameters.json
RunID	A UUID that is assigned to the run and sourced from RunParameters.json
SampleID	A globally unique sample identifier
SampleName	The alphanumeric sample identifier
SampleNumber	The numeric sample identifier
Yield	The number of bases in the sample in gigabases

Occurrences

Occurrences are a set of fields in a sample metrics file that allocate sample performance metrics by specific occurrences of a sample in the run. For example, if a sample appears in both lanes, then Bases2FastqDx lists an occurrence for each lane.

Each occurrence includes the identifiers Lane and Expected Sequence, and reports the following performance metrics:

Metric	Value
BaseComposition	Counts for each A, C, G, T, and N base
CustomMetadata	Custom metadata that is specified in the run manifest, if applicable
MeanReadLength	The average read length after adapter trimming
NumPolonies	The total number of polonies that are assigned to the sample
NumPoloniesBeforeTrimming	The number of polonies that are assigned to a sample before adapter trimming
PercentMismatch	The percentage of polonies that are assigned to a sample with a mismatch
PercentQ30	The percentage of ≥ Q30 Q scores for the specific occurrence of a sample. This includes assigned reads and excludes filtered reads and no calls.
PercentQ40	The percentage of ≥ Q40 Q scores for the specific occurrence of a sample. This includes assigned reads and excludes filtered reads and no calls.
PercentReadsTrimmed	The percentage of reads that Bases2FastqDx trimmed
PerReadGCCountHistogram	A list of counts: the value at index i is the number of reads with i G/C calls
QualityScoreMean	The mean Q score of base calls for the sample and excludes filtered reads and no calls
R1Adapters	The Read 1 adapter sequences that are associated with the lane that the occurrence belongs to
R2Adapters	The Read 2 adapter sequences that are associated with the lane that the occurrence belongs to
RemovedAdapterLengthHistogram	A histogram that shows the number of bases trimmed from an adapter at a given position
Yield	The number of bases in the sample in gigabases