Bioinformatics FAQ and Answers

Currently we offer primary and secondary analyses for all NGS projects, including initial base-calling, demultiplexing, data quality control, and reference genome alignment of NGS reads. We also offer tertiary analyses on a limited basis for R&D projects, which may include de novo assembly, whole genome structural variant analysis, full length transcriptomic splice variant detection, and single cell analysis. For all projects, we insure that every sequence run we deliver meets our high standard for yield, base-call quality, and base alignment percentage and application specific standard metrics that we established.

Coverage requirements vary by application, library protocol, sequencing platform, and project specific considerations. To provide the best approach for your project, a meeting is setup between you and representatives from our sequencing facility in order to make recommendations in sequencing platform, library protocol, and other needs.

For assistance in planning your experiment or to discuss specifics of your project please contact Bao Tran (bao.tran@nih.gov) for bioinformatics consultation please contact Yongmei Zhao (yongmei.zhao@nih.gov).

Please go to the following web links for experimental design best practices:

• ATAC-seq Best Practices: https://informatics.fas.harvard.edu/atac-seq-guidelines.html
• ChIP-seq, RNA-seq, and Exome and Whole Genome-seq: https://bioinformatics.ccr.cancer.gov/ccbr/project-support/experimental-design-best-practices/
• Whole genome sequencing and Structural Variation Detection Best Practices: coming soon
• Single cell RNA-seq: coming soon

We recommend an initial consultation with the CCR-SF Bioinformatics group to discuss data analysis requirements and to establish expectations. It is also important to specify the reference genome version and annotation build for projects with human or mouse genome mapping requirements. For other reference-based sequencing projects, you will need to provide us with the reference sequences (FASTQ file format or weblink).

If you have any questions regarding your preferred data processing options, please contact Yongmei Zhao (yongmei.zhao@nih.gov) or SF Bioinformatics Team via email CCRSF_IFX@nih.gov.

What types of analysis workflows does the CCR-SF use to perform analyses?

We currently provide analyses based on sequencing application type. We have designed and implemented in-house data analysis pipelines that integrate platform/vendor specific data analysis tools with popular open-source software.

Currently available data analysis pipelines:

• Illumina Sequencing
• ATAC-seq
• ChIP-seq
• Exome-seq
• Whole Genome Sequencing for SNVs, CNVs and SVs
• RNA-Seq
• miRNA-Seq
• Whole genome bisulfite-seq
• PacBio Long-read Sequencing
• 16S amplicon
• Iso-seq
• De novo Assembly
• Whole genome sequencing for mutations (SNVs) and structural variant (SV) analysis
• DNA base modification
• HLA Genotyping
• Single Cell Analysis
• Single Cell RNA-seq and CITE-seq
• Single Cell Immune Profiling
• Single Cell Multiome
• Oxford Nanopore Sequencing
• Directed RNA or full-length transcript sequencing
• Adaptive sampling for regions of interest analysis or virus integration sites detection
• Single cell full-length transcriptomic sequencing analysis
• Bionano Optical Genome Mapping
• Whole genome structural variant (SV) analysis and copy number variation (CNV) profiling

For projects using the Illumina sequencing platform, a PDF report containing a summary of the sequencing project (i.e., library and sequencing protocols, sequencing result summary, application-based QC metrics, and software details) and an excel file containing the detailed data analysis results. Depending on the application, you will also receive a html QC report file contains detailed QC statistics and plots for analysis workflows included for that specific application. In addition, you will receive the pass-filtered raw sequence reads in FASTQ format and the reference alignment data in BAM format. BAM files contain base-call and quality score information for all pass-filtered reads, as well as alignment information for reads that have mapped to the reference genome. Additional application specific data files were specified in the deliverable data file types.

For projects using the PacBio sequencing platform, the data delivery choice is driven by the specific needs of the project. For example, when circular consensus processing is performed, the raw subreads bam file, run definition xml files, and the consensus reads (CCS) are included in the data delivery package. If alignment and variant calling are performed, the resulting data are provided within BAM and VCF files. There are also files containing the intermediate results of pipeline processing (such as the read-to-cluster mapping for IsoSeq) are sometimes included. Beyond that, we are happy to deliver any of the files produced by our processing upon request. The content of the data delivery package should be discussed at project definition time.

For standard projects, the deliverable data file types are:

• Sequencing FASTQ/FASTA files
• Alignment BAM files or assembly files
• Data QC statistics reports
• Mapping or variant calling statistics

For projects with secondary and application specific analysis, the deliverable data file types are:

• Exome-seq or WGS Structural Variants Discovery:
• Raw FASTQ files
• Alignment BAM files
• SNP/Indel and structural variant call VCF files
• Sturctural variant call BED file
• Variant annotation files
• QC and variant analysis statistics reports
• RNA-Seq:
• Raw FASTQ files
• STAR-2pass alignment BAM files
• Rsem gene and transcript quantification count matrix files
• QC and RNA analysis statistics reports
• PacBio Iso-seq:
• Raw data: CCS/HiFi reads BAM or FASTQ
• QC and Statistics reports: MultiQC report, Squanti3 report and Kraken contamination check
• Analysis data: high quality clustered isoforms, full length cDNAs, Squanti3 filtered results including BAM, GTF as well as classification table.
• PacBio De novo Assembly:
• Raw data: CCS reads BAM or FASTQ
• QC and Statistics reports: MultiQC report, assembly report and Kraken contamination check
• Analysis data: polished contigs
• PacBio Long Amplicon Sequencing:
• Raw data: CCS reads BAM or FASTQ
• QC and Statistics reports: MultiQC report, Kraken contamination check
• Analysis data: Clustered long amplicon consensus, phasing and variant analysis file
• PacBio WGS Sequencing:
• Raw data: CCS reads BAM or FASTQ
• QC and Statistics reports: MultiQC report, Kraken contamination check
• Analysis data: mapped BAM file, SV VCF file
• PacBio HLA Genotyping:
• Raw data: CCS reads BAM or FASTQ
• QC and Statistics reports: MultiQC report, Kraken contamination check
• Analysis data: mapped BAM files, standard HLA genotyping reports
• Single Cell RNA:
• Cell Ranger output
• Seurat clustering
• SingleR annotations
• Nozzle report
• Single Cell ATAC:
• Cell Ranger output
• Signac clustering
• Single Cell Multiome:
• Cell Ranger output
• Single Cell Immune Profiling:
• Cell Ranger output
• Single Cell Fixed RNA Profiling:
• Cell Ranger output
• Single Cell CNV:
• Cell Ranger DNA output
• Single Cell PIPseq:
• PIPseeker output

Currently we offer primary and secondary analyses for all NGS projects, including initial base-calling, demultiplexing, data quality control, and reference genome alignment of NGS reads. We also offer tertiary analyses on a limited basis for R&D projects, which may include de novo assembly, whole genome structural variant analysis, full length transcriptomic splice variant detection, and single cell analysis. For all projects, we insure that every sequence run we deliver meets our high standard for yield, base-call quality, and base alignment percentage and application specific standard metrics that we established.