Output Files

The following table lists the files that Bases2Fastq outputs.

File	Directory	Description
Bases2Fastq.log	info	Log file that records software events
IndexAssignment.csv	Root	Yield and the number and rate of polonies assigned for each sample and index combination
Metrics.csv	Root	The mismatch rates, percent assigned, and per sample yield for each lane
{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 and the number and rate of polonies assigned for each sample and index combination in a project
{ProjectName}_metrics.csv	Samples/{ProjectName}	The 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	The run manifest for the Bases2Fastq execution
RunManifest.json	Root	Machine-readable copy of the run manifest as a JSON file
RunManifestErrors.json	info	A record of errors in the run manifest
RunParameters.json	Root	A copy of the original run parameters file
RunStats.json	Root	Information on run performance
{SampleName}_{Read}.fastq.gz	Samples/{SampleName} or Samples/{ProjectName}/{SampleName}	The primary output of Bases2Fastq
{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 counts1

^{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. FASTQ files are GZIP-compressed text files that Bases2Fastq names {SampleName}_{Read}.fastq.gz.

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 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 run manifest with no samples or associated index sequences, Bases2Fastq assigns all reads to DefaultSample. The software only produces the FASTQ files DefaultSample_R1.fastq.gz and DefaultSample_R2.fastq.gz.

Sequence Identifiers

A sequence identifier includes the components described in the following table, formatted in one line:

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

Component	Value	Description
@	@	Start to the sequence identifier line
<instrument>	Upper and lowercase letters, integers 0–9, and underscores (_)	Instrument name
<run name>	Upper and lowercase letters, integers 0–9, hyphens (-), and underscores	Run name as defined during run setup
<flow cell ID>	Upper and lowercase letters and integers 0–9	Flow Cell ID from the barcode scan, with the Run ID replacing the Flow Cell ID if no barcode is present
<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 separating 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 backwards compatibility and does not change
<control number>	0	A legacy control number of 0 that exists only for backwards compatibility and does not change
<index sequence>	Varies	A value that depends on the indexing strategy 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 indicates the confidence of a base call based on the Phred scale. 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. Bases2Fastq 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

The HTML QC reports are organized in 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, the report does not display per sample charts.

Bases2Fastq names the QC report for a run {RunName}_QC.html and 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 configured for static binary, complete the following troubleshooting steps.

1. Make sure compatible versions of Python and the necessary packages are installed.
2. Review the error in info/QCReportErrors.txt for the cause, and then use this information to generate the HTML QC report.

Metrics Files

Bases2Fastq reports metrics in different files and formats to support different use cases.

• Metrics.csv offers a high-level overview of yield and assignment metrics, both 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.

Note:

Output files with metrics only report PercentQ50 for runs that use Cloudbreak UQ chemistry. For other types of sequencing chemistry, the JSON files report PercentQ50 values of null, and the CSV files report PercentQ50 values of empty.

Run Metrics (RunStats.json)

A run metrics file, RunStats.json, reports the following performance metrics in a JSON file format. The metrics are specific to the Bases2Fastq execution.

Metric	Value
AnalysisID	The unique identifier that Bases2Fastq generates for the analysis
AnalysisVersion	The current version of Bases2Fastq
AssignedYield	The run yield based on assigned reads in gigabases
FileVersion	The current version of the file format
FlowCellID	A flow cell identifier sourced from RunParameters.json or, if blank, the letter R followed by the RunID value
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 calculated for the run
NumPoloniesBeforeTrimming	The total number of polonies calculated for the run before adapter trimming
PercentAssignedReads	The percentage of reads assigned to a sample
PercentMismatch	The percentage of polonies assigned to a sample with a mismatch
PercentMismatchI1	The percentage of polonies assigned to Index 1 sequences with a mismatch
PercentMismatchI2	The percentage of polonies assigned to Index 2 sequences with a mismatch
PercentQ30	The percentage of ≥ Q30 Q scores for the run, including assigned and unassigned reads
PercentQ40	The percentage of ≥ Q40 Q scores for the run, including assigned and unassigned reads
PercentQ50	The percentage of ≥ Q50 Q scores for the run, including assigned and unassigned reads
PercentReadsTrimmed	The percentage of reads that Bases2Fastq trimmed
PercentUnexpectedIndexPairs	The percentage of all polonies with Index 1 and Index 2 reads that matched different samples1
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 average Q score of base calls for a sample
RemovedAdapterLengthHistogram	A histogram showing the number of bases trimmed from an adapter in a given position
RunName	A text-based run identifier sourced from RunParameters.json
RunID	A universally unique identifier (UUID) assigned to the run and sourced from RunParameters.json
Samples	A list of libraries the run sequenced
SampleStats	The per-sample metrics 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 ({ProjectName}_RunStats.json)

When a run manifest groups samples by project, Bases2Fastq creates JSON project metrics files. Bases2Fastq 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 Bases2Fastq generates for the analysis
AnalysisVersion	The current version of Bases2Fastq
BaseComposition	Counts for each A, C, G, T, and N base
FileVersion	The current version of the file format
FlowCellID	A flow cell identifier sourced from RunParameters.json or, if blank, the letter R followed by the RunID value
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 calculated for the samples in the project
NumPoloniesBeforeTrimming	The total number of polonies calculated for the samples in the project before adapter trimming
PercentMismatch	The percentage of polonies assigned to samples with a mismatch in the project
PercentMismatchI1	The percentage of polonies assigned to Index 1 sequences with a mismatch
PercentMismatchI2	The percentage of polonies assigned to Index 2 sequences with a mismatch
PercentQ30	The percentage of ≥ Q30 Q scores for the project, including assigned and unassigned reads
PercentQ40	The percentage of ≥ Q40 Q scores for the project, including assigned and unassigned reads
PercentQ50	The percentage of ≥ Q50 Q scores for the project, including assigned and unassigned reads
PercentReadsTrimmed	The percentage of reads that Bases2Fastq 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
Reads	A detailed list of per read metrics
RemovedAdapterLengthHistogram	A histogram showing the number of bases trimmed from an adapter in a given position
RunName	A text-based run identifier sourced from RunParameters.json
RunID	A UUID assigned to the run and sourced from RunParameters.json
Samples	A list of libraries sequenced for the project
SampleStats	The per-sample metrics 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 ({SampleName}_stats.json)

Sample metrics files report the following sample-specific performance metrics in the JSON file format. Bases2Fastq names the files {SampleName}_stats.json.

Metric	Value
AnalysisVersion	The current version of Bases2Fastq
BaseComposition	Counts for each A, C, G, T, and N base
ExternalID	An external ID 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 assigned to the sample
NumPoloniesBeforeTrimming	The number of polonies assigned to a sample before adapter trimming
Occurrences	Additional information per occurrence of the sample
PercentMismatch	The percentage of polonies assigned to a sample with mismatch
PercentQ30	The percentage of ≥ Q30 Q scores for the sample
PercentQ40	The percentage of ≥ Q40 Q scores for the sample
PercentQ50	The percentage of ≥ Q50 Q scores for the sample
PercentReadsTrimmed	The percentage of reads that Bases2Fastq 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
RemovedAdapterLengthHistogram	A histogram showing the number of bases trimmed from an adapter in a given position
RunName	A text-based run identifier sourced from RunParameters.json
RunID	A UUID 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, Bases2Fastq 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 specified in the run manifest, if applicable
MeanReadLength	The average read length after adapter trimming
NumPolonies	The total number of polonies assigned to the sample
NumPoloniesBeforeTrimming	The number of polonies assigned to a sample before adapter trimming
Occurrences	The average read length after adapter trimming
PercentMismatch	The percentage of polonies assigned to a sample with mismatch
PercentQ30	The percentage of ≥ Q30 Q scores for the run, including assigned and unassigned reads
PercentQ40	The percentage of ≥ Q40 Q scores for the run, including assigned and unassigned reads
PercentQ50	The percentage of ≥ Q50 Q scores for the run, including assigned and unassigned reads
PercentReadsTrimmed	The percentage of reads that Bases2Fastq 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
R1Adapters	The Read 1 adapter sequences associated with the lane the occurrence belongs to
R2Adapters	The Read 2 adapter sequences associated with the lane the occurrence belongs to
RemovedAdapterLengthHistogram	A histogram showing the number of bases trimmed from an adapter in a given position
Yield	The number of bases in the sample in gigabases