The Samples section includes the
ExternalID columns. When the
Samples section is populated, only
SampleName is required. When unpopulated, Bases2Fastq defaults to an entry that specifies one unindexed sample for each lane.
|SampleName||The name of each library in the run. Each name can include 1–64 case-sensitive alphanumeric characters, hyphens (-), and underscores (_).|
|Index1, Index2||The Index 1 and Index 2 sequences that correspond to each library. Each sequence consists of A, C, G, and T, with a plus sign separating multiple sequences.|
|Lane||The number of a lane to restrict a library to: 1, 2, or 1+2 (default). If using the individually addressable lanes add-on with two library pools, lane 1 refers to the Library well, and lane 2 refers to the AUX well.|
|Project||The name of a project to group samples together. Each name can include case-sensitive alphanumeric characters, hyphens (-), and underscores (_). |
Project names for samples must exactly match to successfully group samples together. Make sure no sample is assigned to more than one project. Bases2Fastq assigns any samples missing a project to a default project named
|ExternalID||An alternative sample name or other external identifier to associate a library with. Each identifier can include 1–64 alphanumeric characters, hyphens, and underscores.|
Recording Index Sequences
The index sequences recorded for an experimental library depend on the library prep workflow and
--force-index-orientation argument in Bases2Fastq. When the argument is used, Bases2Fastq uses the orientation recorded in the run manifest. By default, Bases2Fastq detects and uses the correct index sequence orientation regardless of the orientation recorded in the run manifest.
Sample to Index Mapping
A repeated sample name links the specified index sequences to each other, so you must consistently capitalize sample names. Single or dual indexes can be specified as follows.
- If you specify multiple indexes for
Index 2in the same row, Bases2Fastq uses all possible pairwise combinations.
- In the
SampleNamecolumn, enter the name of each library in the run.
- In the
Index2columns, enter the index sequences for each library in the same orientation.
- For unindexed libraries, leave the
- For single-indexed libraries, leave the
- For unindexed libraries, leave the
When recording sequences in the
Index2 columns, use the same orientation for each sequence in a column. Entering the wrong sequences or mixing orientations affects index assignment.
- For the Adept Workflow, enter the third-party index sequences.
- If orientation detection is enabled in Bases2Fastq, enter the third-party index sequences as found in documentation.
- If orientation detection is disabled in Bases2Fastq, enter the third-party index sequences in the correct orientation. For guidance, see Read Orientation.
Index Sequence Selection
- For the Elevate Workflow, enter the Elevate Version 1 Index Sequences or Elevate Cloudbreak™ Index Sequences.
- For the LoopSeq for AVITI, enter the LoopSeq for AVITI Index Sequences.
- If a run includes a PhiX spike-in, enter the applicable sample names and index sequences listed in PhiX Control Library Index Sequences. Each PhiX Control library includes four index pairs with defined names and sequences.
The Adept Workflow depends on a third-party kit, so an Adept library has P5 and P7 sides. The P5 and P7 sequences are not Element components. In contrast, the Elevate Workflow uses Element indexes and adapters to prepare libraries with SP5 and SP27 sides.
AVITI OS v2.0.0 or later sequences the library and the complementary sequence, including any indexes. Index 1, Index 2, and Read 1 occur in order on the P5 or SP5 side. The run then performs Read 2 on the P7 or SP27 side.
Figure 2: Sequencing Adept and Elevate libraries
Custom Sample-Level Metadata
An optional addition to the Samples section, custom columns specify additional key-value metadata for samples. Custom columns must meet the following requirements:
- Each custom column name is unique, including case-sensitive variations.
- A custom column cannot have the same name as a required column.
- When a sample name is repeated, each corresponding value in the custom column must match exactly.
- The key character limit is 1–64. Valid characters include lower- and upper-case letters, numbers, hyphens, and underscores.
- The value character limit is 0–255 ASCII characters.
Example Samples Usage
Using Individually Addressable Lanes with Projects
- Use the Individually Addressable Lanes add-on with two library pools and associate samples with projects Library_Pool_1 or Library_Pool_2.
Adding Custom Metadata
- Add a custom column, Custom_Metadata, with metadata Sample_1_metadata, Sample_2_metadata, and Sample_3_metadata.
Defining Index Sequences
- Associate a sample named ID1 with the Index 1 sequences AAAA and TTTT. You can organize the index sequences within a single column and use a plus sign (+) to combine them. You can also list the index sequences in individual sample rows.
- Associate a sample named ID1 to a combined Index 1 and Index 2 sequence, AAAATTTT.
- Associate a sample named ID1 to the combined Index 1 and Index 2 sequences AAAATTTT and CCCCGGGG.
- Associate a sample named ID1 to the combined Index 1 and Index 2 sequences AAAATTTT, AAAAGGGG, CCCCTTTT, and CCCCGGGG.