Skip to content

Sequencing Level 2

A Sequencing Level 2 entry documents sequencing data that has been aligned to a reference genome. This level builds directly on a Sequencing Level 1 file and adds alignment-specific metrics, such as aligned reads, deduplicated reads, trimmed reads, mapping quality (MapQ30), and the genomic reference and software version used to perform the alignment.

Level 2 entries make explicit the connection between a raw sequencing file and the processed alignment derived from it, while also capturing the tools and parameters that produced the alignment, which is essential for evaluating data quality and reproducing analyses.

Why You Should Contribute Sequencing Level 2 Entries

Contributing Sequencing Level 2 entries ensures that alignment outputs are traceable to their raw source files and are documented with the quality metrics and software versions needed for others to assess, reproduce, or build on the analysis.

Who Should Be Contributing Sequencing Level 2 Entries?

  1. Computational Genomics Analysts – Record alignment metrics and software versions immediately after processing pipelines complete.
  2. Sequencing Core Staff – Confirm that aligned outputs are correctly linked back to the raw files and biospecimens they originated from.
  3. Bioinformatics Pipeline Developers – Document the genomic reference and workflow versions used, supporting reproducibility across pipeline updates.
  4. Data Managers – Maintain consistent, portal-ready metadata for aligned sequencing outputs shared through the CCKP.

Download Template

You can download the SequencingLevel2 CSV template to streamline data entry.

Full Field Reference

Below is the full field reference table with attributes and their descriptions.

Attribute Description Required Column Type Format Regex Pattern Standard Terms Examples
SequencingLevel2_id Unique row identifier, used as a primary key for record updates. This should be equivalent to the file Synapse Id unless otherwise indicated. True string None ^syn\d{7,8}$ None
SequencingLevel1 Key Unique SequencingLevel1_id foreign key(s) that link metadata entries as part of the same Dataset. Please provide multiple values as a comma-separate list. False string None None None
Filename The path of a file in Synapse, relative to the project. The file associated with the path will be annotated with attributes contained in this sheet. False string None None None
Biospecimen Key Unique Biospecimen_id foreign key(s) that link metadata entries as part of the same collection. Please provide multiple values as a comma-separated list. False string None -B\d{1,9} None
Study Key The unique Study_id foreign keys associated with the resource, found in the grant Study information. Used to group the resource with other components. Please provide multiple values as a comma-separated list. False string None None None
DatasetView Key Unique DatasetView_id foreign key(s) that link metadata entries as part of the same collection. Please provide multiple values as a comma-separated list. False string None None None
File Alias A string identifier associated with the file. Must be unique. Can be the repository accesssion number (e.g., Synapse ID, GEO identifier such as GSE12345). No Greek Letters or DOIs. True string None None None
File Description Description of the file. False string None None None
File Design The overall design of the dataset or file, including a batch identifier, if applicable. False string None None None
File Level The processing level the file can be mapped to. True string None None View
File Assay The assay the file is representative of. True string None None View
File Species The species the data was collected on. True string None None View
File Url The url of where the file is stored. True string uri None None
File Format The format of the file described by this entry. True string None None View
File Data Use Codes DUO code - A data item that is used to indicate consent permissions for datasets and/or materials, and relates to the purposes for which datasets and/or material might be removed, stored or used. Available DUO code definitions can be found here: https://mc2-center.github.io/data-models/valid_values/study/#attribute-study-data-use-codes False string_list None None None
File Longitudinal Group A label that can be used to identify groups of files from the same longitudinal/time-resolved experiment False string None None None
File Longitudinal Event Type The type of event to which File Longitudinal Total Time Elapsed is related False string None None View
File Longitudinal Sequence Identifier The order in which this file was collected with respect to the longitudinal experiment (e.g., 1, 2, etc.). Integer. False number None None None
File Longitudinal Time Elapsed Unit The unit of time associated with Sequential and Total Time Elapsed attributes. False string None None None
File Longitudinal Total Time Elapsed The total time elapsed between the first and current files contained this longitudinal group. False number None None None
NGS Library Strategy The assay associated with the nucleic acid library (e.g., RNA-Seq, ChIP-Seq) True string None None View
NGS Library Source Material The type of source material being sequenced. True string None None View
NGS Library Source Molecule The source of nucleic acids represented in the sequencing library. True string None None View
NGS Library Selection Method How nucleic acid molecules are isolated. False string None None View
NGS Library Layout Text description of the library layout, one of Paired-end or Single. True string None None View
NGS Sequencing Platform A platform is an object aggregate that is the set of instruments and software needed to perform a process [OBI_0000050]. Specific model of the sequencing instrument. True string None None View
NGS Sequencing Design Description Free-form description of the methods used to create the sequencing library; a brief 'materials and methods' section. True string None None None
NGS Raw Reads Reads not yet analyzed in any way to be used for data analysis. The number of reads that pass filter from the flow cell represented in the FASTQ file. False number None None None
NGS Stitched Reads Represents consensus from the overlapping sequence of read 1 and 2. This is a % of the aligned reads that were overlapped and consensus confirmed, usually upward of 80% but less in terms of number of reads than aligned reads False number None None None
NGS Aligned Reads Is a sequence that has been aligned to a gene/probe. Typically these reads can number from the hundreds of thousands to tens of millions. In GeoMx alignment is via mapping the RTS ID to a white list of sequences that represent targets. False number None None None
NGS Deduplicated Reads Is the replacement of blocks of duplicate data with a Virtual Index Pointer linking the new sub-block to the existing block of data in a duplicate repository. This is used to reduce the amount of space need to store the data. False number None None None
NGS Trimmed Reads Number of the reads remaining after trimming has been applied. False number None None None
NGS MapQ30 Number of reads with Quality >= 30. False number None None None
NGS Unique Bases Count of unique basecalls present in the data. False number None None None
NGS Read Length The average length of the sequencing reads. Can be integer, null True number None None None
NGS Sequencing Coverage Depth of coverage on assembly used. Found by (Unique Aligned Basecalls)/(Reference Length) False number None None None
Genomic Reference Exact version of the human genome reference used in the alignment of reads (e.g. GCF_000001405.39) False string None None None
Software and Version Name of software used to generate the information contained in the file. String False string None None None