Google DeepOmics

Summary

This workflow is designed to help the user determine differential gene abundances and differential expression between two or more groups of samples. The user will provide as input a folder containing all the read files needed for analysis and a sequencing file relating sample IDs to attributes. The user will receive as output differential gene and transcript abundance analysis files and comparison files between the two or more samples.

Methods

This analysis was performed using the Expression Analysis in RNASeq workflow on the Form Bio platform. Reads are trimmed using TrimGalore [1], to remove low quality (qual < 25) ends of reads and remove reads < 35bp. Trimmed reads are aligned to a reference genome using STAR [2] (default) or HiSAT2 [3]. Duplicate reads can optionally be marked using Picard MarkDuplicates. BAMs from the same sample generated by multiple runs are merged using Samtools [4]. The abundance of transcripts and genes are assessed using FeatureCount to generate raw gene counts [5], StringTie to generate FPKM [6] and Salmon to generate raw transcript counts [7]. Sample comparisons and differential gene/transcript expression analysis are performed using EdgeR [8], DESeq2 [9] and IsoformSwitchAnalyzeR [10].

Summary

This workflow is designed to help the user determine differentially expressed genes using abundance counts generated by previous workflow analysis. The user will provide a folder containing output files from previous analyses. The workflow will perform the statistical analysis again with a different composition of samples and output the results of this analysis.

Methods

Sample comparisons and differential gene/transcript expression analysis are performed using EdgeR [8], DESeq2 [9] and IsoformSwitchAnalyzeR [10].

Summary

This workflow is designed to help the user determine differential gene abundances and differential expression between two or more groups of human tumor samples. The user will provide as input a folder containing all read files needed for analysis and a sequencing file relating sample IDs to attributes. The user will receive as output differential gene and transcript abundance analysis files and comparison files between the two or more samples.

Methods

This analysis was performed using the Expression Analysis in RNASeq workflow on the Form Bio platform. Reads are trimmed using TrimGalore [1], to remove low quality (qual < 25) ends of reads and remove reads < 35bp. Trimmed reads are aligned to a reference genome using STAR [2] (default) or HiSAT2 [3]. Duplicate reads can optionally be marked using Picard MarkDuplicates. BAMs from the same sample generated by multiple runs are merged using Samtools [4]. The abundance of transcripts and genes are assessed using FeatureCount to generate raw gene counts [5], StringTie to generate FPKM [6] and Salmon to generate raw transcript counts [7]. Sample comparisons and differential gene/transcript expression analysis are performed using EdgeR [8], DESeq2 [9] and IsoformSwitchAnalyzeR [10]. Gene fusion events are detected using Star-Fusion [11]. Exon-skipping events are detected using RegTools [12].

Summary

This workflow is designed to help the user determine germline variants in diploid DNA. The user will provide input FastQ files containing the diploid DNA to be analyzed, and will recieve as output a summary of germline variants in the DNA compared to the chosen reference genome.

Methods

This analysis was performed using the Germline Variant Analysis workflow on the Form Bio platform. When the datatype is “Germline (Diploid)”, this workflow determines genetic variants including SNVs, insertions and deletions of high-quality NGS data when compared to a reference genome. Reads are trimmed using TrimGalore [1] or FastP [2], to remove low quality (qual < 25) ends of reads and remove reads < 35bp. These default value can be changed by the user. This workflow can be run with native open-source tools (NOST), Sentieon or with Parabricks.

With NOST and Sentieon, trimmed reads are aligned to a reference genome using BWA-MeM [3], Minimap2 [4] or Winnowmap [5] depending on data type. Duplicate reads can optionally be marked using Picard MarkDuplicates [6]. BAMs from the same sample generated by multiple runs are merged using Samtools [7]. Alignment qualtity is assessed using FastQC [8], Samtools [7], Bedtools [9] and Qualimap [10]. Variants can be detected with joint calling using Freebayes [11], Samtools/Bcftools [12], DNAScope and GATK4 [13].

With Parabricks, trimmed reads are aligned, duplicate reads are marked and alignment quality is accessed using fq2bam. Quality metrics are summarized with MultiQC. Variants can be detected with GATK [13] and DeepVariant [14] to produce gVCF files. Genotyping of gVCF files is determined using GLNexus [15]. Variants effects are determined using SNPEff [16].

Summary

This workflow is designed to help the user determine variants in ancient DNA. The user will provide input FastQ files containing the DNA to be analyzed, and will recieve as output a summary of variants in the DNA compared to the chosen reference genome.

Methods

This analysis was performed using the Germline Variant Analysis workflow on the Form Bio platform. When the datatype is “Ancient”,this workflow can be used to determine genetic variants of high quality NGS data in your project compared to a supported reference genome. Reads are trimmed using AdapterRemoval [17], FastP [2], or TrimGalore [1], to remove low quality (qual < 25) ends of reads and remove reads < 35bp. These default value can be changed by the user. Contaminates are detected using Kraken [18] with confidence of 0.8 using Kraken’s precompiled database or a custom database where the human genome has been removed. Unclassified trimmed reads are aligned to a reference genome using BWA MEM [3] or BWA Aln (with seed of 16,500, maximum edit distance of 0.01 and maximum gap opens of 2). BAMs from the same library generated by multiple runs are merged using Samtools [7]. Duplicate reads from the the same library can optionally be marked using PaleoMIX [19] or Picard MarkDuplicates [6]. BAMs from the same sample generated by multiple libraries are merged using Samtools [7]. Base recalibration is done using mapdamage2 [20]. Alignment quality is assessed using QualiMap [21], DamageProfiler [22], and MultiQC [23]. Germline variants can be detected using Freebayes [11], Samtools/Bcftools [12] and GATK4 [13]. In order to increase the speed of analysis, the Parabricks (requires GPUs) or Sentieon optimized versions of these algorithms are used for BWA Mem and GATK4. Genotyping of GVCF files is determined using GLnexus [15]. Variant effects are determined usng SNPEff [16].

Summary

This workflow is designed to help the user determine germline variants in small or synthetic genomes with an option to provide the custom genome sequence. The user will provide input FastQ files containing the DNA to be analyzed, and will recieve as output a summary of variants in the DNA compared to the chosen reference genome. It is assumed that Small genomes are haploid.

Methods

This analysis was performed using the Germline Variant Analysis workflow on the Form Bio platform. When data type is a “Small Genome”, this workflow can be used to determine genetic variants of high quality NGS data in your project compared to a supported reference genome. Reads are trimmed using TrimGalore [1]. Trimmed reads are aligned to a reference genome using BWA MEM [3]. Duplicate reads are marked using Picard MarkDuplicates [6]. Germline variants can be detected using Samtools/Bcftools [12]. In order to increase the speed of analysis, the Parabricks or Sentieon optimized versions of these algorithms can be used for BWA MEM. Variant effects are determined usng SNPEff [16] if the genome is provided by the platform. For SARS-CoV-2, genome sequences of samples are determined using BCFTools [12] and lineage classification is determined using PANGOLIN [24].

Summary

This workflow is designed to help the user determine variants in somatic DNA. The user will provide input FastQ files containing the DNA to be analyzed, and will receive as output a summary of variants in the DNA compared to the chosen reference genome.

Methods

When data type is “Somatic”, this workflow can be used to determine genetic variants of tumor NGS data compared to a supported reference genome. When a normal sample is provided, specialized somatic variant calling methods will be applied and allow users to filter germline variants from the resulting VCF files. Reads are trimmed using TTrimGalore [1] or FastP [2], to remove low quality (qual < 25) ends of reads and remove reads < 35bp. These default value can be changed by the user. Trimmed reads are aligned to a reference genome using BWA MEM [3], Minimap2 [4] or Winnowmap [5] depending on data type. Duplicates reads are marked using Picard MarkDuplicates [6]; if provided by Library. BAMs from the same sample generated by multiple runs are merged using Samtools [7]. Alignment quality is assessed using FastQC [8], Samtools [7], Bedtools [9] and Qualimap [10]. Quality reports are produced by MultiQC. Variant effects are determined using SNPEff [16]. Somatic variants can be detected in somatic or tumor-only mode using Strelka2 [25], Freebayes [11], DeepSomatic and MuTect2 [26], TNScope. In order to increase the speed of analysis, the Parabricks or Sentieon optimized versions of these algorithms are used for BWA Mem and GATK4. When a matched normal sample is present, tumor/normal germline SNP matching is confirmed using NGSCheckMate [27] and microsatellite stability is assessed using MSI-Sensor [28].

Summary

This workflow is designed to help create draft genome assemblies from short-read sequencing data, such as Illumina or WGS. The user will provide as input a directory of sequencing files as well as optional OMNI-C and/or HiC data for polishing. The user will receive as output a draft genome assembly.

Methods

This analysis was performed using the Genome Assembly workflow on the Form Bio platform. Short reads are first interleaved with bbmap [1] if paired-end and not yet interleaved before combining them into a singular file. Once reads are consolidated, they are assembled with Spades [2], MetaSpades [3], Megahit [4], and/or Skesa [5]. Finally, the created assemblies are evaluated with Quast [6], Busco [7], and/or Merqury [8].

Summary

This workflow is designed to help the user create draft genome assemblies from long-read sequencing data, such as PacBio or ONT data. The user will provide as input a directory of sequencing files as well as optional OMNI-C and/or HiC data for polishing. The user will receive as output a draft genome assembly.

Methods

This analysis was performed using the Genome Assembly workflow on the Form Bio platform. This workflow first performs basecalling if desired. This basecalling can be ONT basecalling when supplied with Fast5/Pod5 files either using Dorado or Bonito. Alternatively, if input data is PacBio subread uBAMs or the sequencer run folder, consensus contig reads are created using circular consensus sequencing (CCS) [9]. Optionally DeepConsensus [10] can be used to improve basecalls. If Bonito is run modbam2bed can optionally be run to create a methyl BED file. Using the provided input FastQ files or ONT basecalls a draft assembly is created with Flye [11], Shasta [12], Verkko [13], and/or Hifiasm [14]. Once draft assemblies are made, they are then polished with polishers running in the following order: racon [15], medaka consensus, pilon [16], juicer [17] + 3ddna [18], TGS Gap Closer [19], DeepPolisher [20], Ragtag Scaffold [21], Ragtag Patch [21], Gapless [22]. After polishing, optional purging can be run with the Purge Dups workflow using minimap2 [23] and small contigs can be filtered out with Seqtk. Between all steps in the workflow, the created assemblies are evaluated with Quast[6], Busco [24], and/or Merqury [8].