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11 KiB
Markdown
226 lines
11 KiB
Markdown
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layout: page
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title: HISAT-3N
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permalink: /hisat-3n/
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order: 4
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share: false
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---
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HISAT-3N
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============
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Overview
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-----------------
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**HISAT-3N** (hierarchical indexing for spliced alignment of transcripts - 3 nucleotides)
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is designed for nucleotide conversion sequencing technologies and implemented based on HISAT2.
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There are two strategies for HISAT-3N to align nuleotide conversion sequencing reads: *standard mode* and *repeat mode*.
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The standard mode align reads with standard-3N index only, so it is fast and require small memory (~9GB for human genome alignment).
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The repeat mode align reads with both standard-3N index and repeat-3N index, then output 1,000 alignment result (the output number can be adjust by `--repeat-limit`).
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The repeat mode can align nucleotide conversion reads more accurately,
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and it is only 10% slower than the standard mode with tiny more memory (repeat mode use about ~10.5GB) usage than standard mode.
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HISAT-3N is developed based on [HISAT2], which is particularly optimized for RNA sequencing technology.
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HISAT-3N can be used for any base-converted sequencing reads include [BS-seq], [SLAM-seq], [TAB-seq], [oxBS-seq], [TAPS], [scBS-seq], and [scSLAM-seq],.
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[HISAT2]:https://github.com/DaehwanKimLab/hisat2
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[BS-seq]: https://en.wikipedia.org/wiki/Bisulfite_sequencing
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[SLAM-seq]: https://www.nature.com/articles/nmeth.4435
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[scBS-seq]: https://www.nature.com/articles/nmeth.3035
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[scSLAM-seq]: https://www.nature.com/articles/s41586-019-1369-y
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[TAPS]: https://www.nature.com/articles/s41587-019-0041-2
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[TAB-seq]: https://doi.org/10.1016/j.cell.2012.04.027
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[oxBS-seq]: https://science.sciencemag.org/content/336/6083/934
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Getting started
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============
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HISAT-3N requires a 64-bit computer running either Linux or Mac OS X and at least 16 GB of RAM.
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A few notes:
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1. The repeat 3N index building process requires 256 GB of RAM.
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2. The standard 3N index building requires no more than 16 GB of RAM.
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3. The alignment process with either standard or repeat index requires no more than 16 GB of RAM.
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4. [SAMtools] is required to sort SAM file for hisat-3n-table.
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Install
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------------
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git clone https://github.com/DaehwanKimLab/hisat2.git
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cd hisat2
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git checkout -b hisat-3n origin/hisat-3n
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make
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Make sure that you are in the `hisat-3n` branch
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Build a 3N index with `hisat-3n-build`
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-----------
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`hisat-3n-build` builds a 3N-index, which contains two hisat2 indexes, from a set of DNA sequences. For standard 3N-index,
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each index contains 16 files with suffix `.3n.*.*.ht2`.
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For repeat 3N-index, there are 16 more files in addition to the standard 3N-index, and they have the suffix
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`.3n.*.rep.*.ht2`.
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These files constitute the hisat-3n index and no other file is needed to alignment reads to the reference.
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* Example for standard HISAT-3N index building:
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`hisat-3n-build genome.fa genome`
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* Example for repeat HISAT-3N index building (require 256 GB memory):
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`hisat-3n-build --repeat-index genome.fa genome`
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It is optional to make the graph index and add SNP or spicing site information to the index, to increase the alignment accuracy.
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for more detail, please check the [HISAT2 manual].
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[HISAT2 manual]:https://daehwankimlab.github.io/hisat2/manual/
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# Standard HISAT-3N integrated index with SNP information
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hisat-3n-build --exons genome.exon genome.fa genome
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# Standard HISAT-3N integrated index with splicing site information
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hisat-3n-build --ss genome.ss genome.fa genome
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# Repeat HISAT-3N integrated index with SNP information
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hisat-3n-build --repeat-index --exons genome.exon genome.fa genome
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# Repeat HISAT-3N integrated index with splicing site information
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hisat-3n-build --repeat-index --ss genome.ss genome.fa genome
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Alignment with `hisat-3n`
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------------
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After we build the HISAT-3N index, you are ready to use `hisat-3n` for alignment.
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HISAT-3N uses the HISAT2 argument but has some extra arguments. Please check [HISAT2 manual] for more detail.
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For human genome reference, HISAT-3N requires about 9GB for alignment with standard 3N-index and 10.5 GB for repeat 3N-index.
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* `--base-change <chr1,chr2>`
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Provide which base is converted in the sequencing process to another base. Please enter
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2 letters separated by ',' for this argument. The first letter(chr1) should be the converted base, the second letter(chr2) should be
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the converted to base. For example, during slam-seq, some 'T' is converted to 'C',
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please enter `--base-change T,C`. During bisulfite-seq, some 'C' is converted to 'T', please enter `--base-change C,T`.
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If you want to align non-converted reads to the regular HISAT2 index, do not use this option.
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* `--index/-x <hisat-3n-idx>`
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The index for HISAT-3N. The basename is the name of the index files up to but not including the suffix `.3n.*.*.ht2` / etc.
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For example, you build your index with basename 'genome' by HISAT-3N-build, please enter `--index genome`.
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* `--repeat-limit <int>`
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You can set up the number of alignment will be check for each repeat alignment. You may increase the number to let hisat-3n
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output more, if a read has multiple mapping. We suggest the repeat limit number for paired-end reads alignment is no more
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than 1,000,000. default: 1000.
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* `--unique-only`
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Only output uniquely aligned reads.
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#### Examples:
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* Single-end slam-seq reads (T to C conversion) alignment with standard 3N-index:
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`hisat-3n --index genome -f -U read.fa -S alignment_result.sam --base-change T,C`
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* Paired-end bisulfite-seq reads (C to T conversion) alignment with repeat 3N-index:
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`hisat-3n --index genome -f -1 read_1.fa -2 read_2.fa -S alignment_result.sam --base-change C,T`
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* Single-end TAPS reads (have C to T conversion) alignment with repeat 3N-index and only output unique aligned result:
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`hisat-3n --index genome -q -U read.fq -S alignment_result.sam --base-change C,T --unique`
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#### Extra SAM tags generated by HISAT-3N:
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* `Yf:i:<N>`: Number of conversions are detected in the read.
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* `YZ:A:<A>`: The value `+` or `–` indicate the read is mapped to REF-3N (`+`) or REF-RC-3N (`-`).
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Generate a 3N-conversion-table with `hisat-3n-table`
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------------
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### Preparation
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To generate 3N-conversion-table, users need to sort the SAM file which generated by `hisat-3n`.
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[SAMtools] is required for this sorting process.
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Use `samtools sort` to convert the SAM file to a sorted SAM file.
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samtools sort alignment_result.sam -o sorted_alignment_result.sam -O sam
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Generate 3N-conversion-table with `hisat-3n-table`:
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### Usage
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hisat-3n-table [options]* --alignments <alignmentFile> --ref <refFile> --output-name <outputFile> --base-change <char1,char2>
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#### Main arguments
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* `--alignments <alignmentFile>`
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SORTED SAM file. Please enter `-` for standard input.
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* `--ref <refFile>`
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The reference genome file (FASTA format) for generating HISAT-3N index.
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* `--output-name <outputFile>`
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Filename to write 3N-conversion-table (tsv format) to.
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* `--base-change <char1,char2>`
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The base-change rule. User should enter the exact same `--base-change` arguments in hisat-3n.
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For example, please enter `--base-change C,T` for bisulfite sequencing reads.
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#### Input options
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* `-u/--unique-only`
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Only count the unique aligned reads into 3N-conversion-table.
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* `-m/--multiple-only`
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Only count the multiple aligned reads into 3N-conversion-table.
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* `-c/--CG-only`
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Only count the CpG island in reference genome. This option is designed for bisulfite sequencing reads.
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* `-p/--threads <int>`
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Launch `int` parallel threads (default: 1) for table building.
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* `-h/--help`
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Print usage information and quit.
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#### Examples:
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* Generate 3N conversion table for bisulfite sequencing data:
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`hisat-3n-table -p 16 --alignments sorted_alignment_result.sam --ref genome.fa --output-name output.tsv --base-change C,T`
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* Generate 3N-conversion-table for TAPS data and only count base in CpG island and uniquely aligned:
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`hisat-3n-table -p 16 --alignments sorted_alignment_result.sam --ref genome.fa --output-name output.tsv --base-change C,T --CG-only --unique-only`
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* Generate 3N conversion table for bisulfite sequencing data from sorted BAM file:
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`samtools view -h sorted_alignment_result.bam | hisat-3n-table --ref genome.fa --alignments - --output-name output.tsv --base-change C,T`
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* Generate 3N conversion table for bisulfite sequencing data from unsorted BAM file:
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`samtools sort alignment_result.bam -O sam | hisat-3n-table --ref genome.fa --alignments - --output-name output.tsv --base-change C,T`
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#### Note:
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There are 7 columns in the 3N-conversion-table:
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1. `ref`: the chromosome name.
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2. `pos`: 1-based position in ref.
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3. `strand`: '+' for forward strand. '-' for reverse strand.
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4. `convertedBaseQualities`: the qualities for converted base in read-level measurement. Length of this string is equal to
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the number of converted Base in read-level measurement.
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5. `convertedBaseCount`: number of distinct read positions where converted base in read-level measurements were found.
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this number should equal to the length of convertedBaseQualities.
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6. `unconvertedBaseQualities`: the qualities for unconverted base in read-level measurement. Length of this string is equal to
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the number of unconverted Base in read-level measurement.
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7. `unconvertedBaseCount`: number of distinct read positions where unconverted base in read-level measurements were found.
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this number should equal to the length of unconvertedBaseQualities.
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##### Sample 3N-conversion-table:
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ref pos strand convertedBaseQualities convertedBaseCount unconvertedBaseQualities unconvertedBaseCount
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1 11874 + FFFFFB<BF<F 11 0
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1 11877 - FFFFFF< 7 0
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1 11878 + FFFBB//F/BB 11 0
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1 11879 + 0 FFFBB//FB/ 10
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1 11880 - F 1 FFFF/ 5
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[SAMtools]: http://samtools.sourceforge.net
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Publication
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============
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* HISAT-3N paper
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Zhang, Y., Park, C., Bennett, C., Thornton, M., & Kim, D. (2021). [Rapid and accurate alignment of nucleotide conversion sequencing reads with HISAT-3N](https://doi.org/10.1101/gr.275193.120). Genome research, gr.275193.120. Advance online publication.
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* HIAST2 paper
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Kim, D., Paggi, J.M., Park, C. _et al._ [Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype](https://doi.org/10.1038/s41587-019-0201-4). _Nat Biotechnol_ **37**, 907–915 (2019)
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