Sample Sheet

A sample sheet (SampleSheet.csv) records information about samples, the corresponding indexes, and other information that dictates the behavior of the software. The default location of the sample sheet is the root output folder. To specify any CSV file in any location, use the command --sample-sheet. When a sample sheet does not exist in the default location and no sample sheet is specified in the command line, the software produces an error.

BCL Convert supports both sample sheets v1 and v2. The following table displays the different supported options for v1 and v2.

Sample Sheet v1

Sample Sheet v2

Supports both [Settings] and [settings]. Neither are required.

Supports only [BCLConvert_Settings]. Required.

Unrecognized settings trigger a warning.

Unrecognized settings produce an error and analysis aborts.

The software uses the settings section of the sample sheet to specify adapter trimming, cycle, UMI, and index options.

Adapter Trimming Specifications

Setting

Description

Default

AdapterRead1

The sequence of the Read 1 adapter to be masked or trimmed.

To trim multiple adapters, separate the sequences with a plus sign (+) to indicate independent adapters that must be independently assessed for masking or trimming for each read.

Allowed characters: A, T, C, G.

Not applicable

AdapterRead2

The sequence of the Read 2 adapter to be masked or trimmed.

To trim multiple adapters, separate the sequences with a plus sign (+) to indicate independent adapters that must be independently assessed for masking or trimming for each read.

Allowed characters: A, T, C, G.

Not applicable

AdapterBehavior

Defines whether the software masks or trims Read 1 and/or Read 2 adapter sequence(s). When AdapterRead1 or AdapterRead2 is not specified, this setting cannot be specified.

• mask—The software masks the identified Read 1 and/or Read 2 sequence(s) with N.
• trim—The software trims the identified Read 1 and/or Read 2 sequence(s)

trim

AdapterStringency

The minimum match rate that triggers masking or trimming. This value is calculated as MatchCount / (MatchCount+MismatchCount). Accepted values are 0.5–1. The default value of 0.9 indicates that only reads with ≥ 90% sequence identity with the adapter are trimmed.

0.9

MinimumAdapterOverlap

Do not trim any bases unless the adapter matches are greater than or equal to the number of bases specified. At least one AdapterRead1 or AdapterRead2 must be specified to use MinimumAdapterOverlap.

Allowed characters: 1, 2, 3.

1

 

Cycle, UMI, and Tile Specifications

Setting

Description

Default

BarcodeMismatchesIndex1

The number of mismatches allowed for index1. Accepted values are 0, 1, or 2.

1

BarcodeMismatchesIndex2

The number of mismatches allowed for index2. Accepted values are 0, 1, or 2.

1

CreateFastqForIndexReads

Specifies whether software outputs FASTQ files for index reads. If index reads are defined as a UMI, then the software outputs FASTQ files for the UMI (if TrimUMI is also set to 0). At least one Index Read must be specified in the sample sheet.

• 0—FASTQ files are not output for index reads.
• 1—FASTQ files are output for FASTQ reads.

0

MinimumTrimmedReadLength

The minimum read length after adapter trimming. The software trims adapter sequences from reads to the value of this parameter. Bases below the specified value are masked with N.

35

MaskShortReads

The minimum read length containing A, T, C, G values after adapter trimming. Reads with less than the specified number of bases become masked. If the value is less than 22, the default becomes the MinimumTrimmedReadLength.

22

OverrideCycles

Specifies the sequencing and indexing cycles that should be used when processing the data. The following format must be used:

• Must be same number of semicolon-delimited fields in string as sequencing and indexing reads specified in RunInfo.xml.
• Indexing reads are specified with I.
• Sequencing reads are specified with Y. UMI cycles are specified with U.
• Trimmed reads are specified with N.
• The number of cycles specified for each read must sum to the number of cycles specified for that read in the RunInfo.xml.
• Only one Y or I sequence can be specified per read.
Example: Y151;I8;I8;Y151

Use reads as specified in the RunInfo.xml

SoftwareVersion

[Optional] Records the version of BCL Convert intended to be used for conversion. Only supported by sample sheet v2.

Not applicable

TrimUMI

Specifies whether UMI cycles are excluded from FASTQ files. You must specify at least one UMI in the sample sheet when this setting is provided.

• 0—UMI cycles are output to FASTQ files.

• 1— UMI cycles are not output to FASTQfiles.

1

NoLaneSplitting

Consolidate FASTQ files across lanes. Each sample must be provided into the same file on a per-read basis.

• Must be true or false.
• Only allowed when Lane column is excluded from the sample sheet.
• If given, must be equivalent to the --no-lane-splitting command-line parameter.

false

The data section is required. Headers for the data section should be [Data] or [data] for sample sheet v1 and [BCLConvert_Data] for sample sheet v2. BCL Convert uses columns in the Data section to sort samples and index adapters.

Column

Description

Lane

[Optional] When specified, the software generates FASTQ files only for the samples with the specified lane number. Only one valid integer is allowed, as defined by the RunInfo.xml.

Sample_ID

The sample ID.

index

The Index 1 (i7) index adapter sequence.

index2

The Index 2 (i5) Index adapter sequence.

Sample_Project

Optional Can only contain alphanumeric characters, dashes, and underscores. Duplicate data strings with different cases (eg, sampleProject and SampleProject) are not allowed. If these data strings are used, analysis fails. This column is not used unless you are using the command line option --bcl-sampleproject-subdirectories. See Command Line Options for more information on command line options.

BCL Convert does not support the following settings, and new formats must replace their corresponding old formats, when applicable. Manual changes to the sample sheet can be made to the [Settings] section, but the [Data] section must remain unchanged. If any of the obsolete settings are used in the command line or the sample sheet, the software aborts and returns an error. Also note that some obsolete settings that were previously specified on the command line are now correctly specified in the sample sheet.

Adapter Behavior and Specifications

Behavior

Obsolete Settings

New Settings

Designate the adapter sequences for Read 1 and Read 2 and specify the behavior as trim.

(sample sheet)

Adapter, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

OR

TrimAdapter, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

(sample sheet)

AdapterRead1, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

AND

AdapterRead2, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

Designate the same adapter sequence for Read 1 and Read 2 and specify the behavior as mask.

(sample sheet)

MaskAdapter, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

(sample sheet)

AdapterRead1, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

AND

AdapterRead2, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

AND

AdapterBehavior, mask

Designate the adapter sequences for Read 1 and Read 2 and specify the behavior as mask.

(sample sheet)

MaskAdapter, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA OR

MaskAdapterRead2, AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

(sample sheet)

AdapterRead1, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

AND

AdapterRead2, AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

AND

AdapterBehavior, mask

Designate the adapter sequences for Read 1 and Read 2 and specify the behavior as trim. Also specify 0.5 as the adapter stringency.

(sample sheet)

Adapter, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

OR

TrimAdapter, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

(command line)

--adapter-stringency 0.5

(sample sheet)

AdapterRead1, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA

AND

AdapterRead2, AGATCGGAAGAGCACACGTCTGAACTCCAGTCA(sample sheet)

AND

AdapterStringency, 0.5

 

Read Trimming

Behavior

Obsolete Settings

New Settings

Trim the first 7 bases and last 6 bases of Read 1 for a 151 x 8 x 8 x 151 run.

(sample sheet)

Read1StartFromCycle, 8

Read1EndWithCycle, 145

(sample sheet)

N7Y137N6;I8;I8;Y151

 

UMI Specification

 

Obsolete Settings

New Settings

Designate the first 8 cycles of Read 1 and Read 2 as UMIs and trim the trailing base for a 151 x 8 x 8 x 151 run.

(sample sheet)

Read1UMIStartFromCycle, 1

Read1UMILength, 8

Read1StartFromCycle, 10

Read2UMIStartFromCycle, 1

Read2UMILength, 8

Read2StartFromCycle, 10

(sample sheet)

U8N1Y142;I8;I8;U8N1Y142

 

Barcode Mismatches

Behavior

Obsolete Command Line Settings

New Sample Sheet Settings

Allow 1 mismatch in the i7 index sequence and 1 mismatch i5 index sequence.

--barcode-mismatches 1

OR

--barcode-mismatches 1,1

BarcodeMismatchesIndex1, 1

AND

BarcodeMismatchesIndex2, 1

Allow 2 mismatches in the i7 index sequence and 2 mismatches in the i5 index sequence.

--barcode-mismatches 2

OR

--barcode-mismatches 2,2

BarcodeMismatchesIndex1, 2

AND

BarcodeMismatchesIndex2, 2

 

Masking of Trimmed Reads

Behavior

Obsolete Command Line Settings

New Sample Sheet Settings

Make sure that all trimmed reads are at least 10 base pairs long after adapter trimming by appending Ns to any read shorter than 10 base pairs.

--minimium-trimmed-read-length 10

MinimumTrimmedReadLength, 10

Make sure that all trimmed reads below 5 base pairs long are masked with Ns.

--mask-short-adapter-reads, 5

MaskShortReads, 5