HISAT2 is a fast and sensitive alignment program for mapping next-generation sequencing reads (whole-genome, transcriptome, and exome sequencing data) against the general human population (as well as against a single reference genome). Based on GCSA (an extension of BWT for a graph), we designed and implemented a graph FM index (GFM), an original approach and its first implementation to the best of our knowledge. In addition to using one global GFM index that represents general population, HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome (each index representing a genomic region of 56 Kbp, with 55,000 indexes needed to cover human population). These small indexes (called local indexes) combined with several alignment strategies enable effective alignment of sequencing reads. This new indexing scheme is called Hierarchical Graph FM index (HGFM). We have developed HISAT 2 based on the HISAT and Bowtie2 implementations. HISAT2 outputs alignments in SAM format, enabling interoperation with a large number of other tools (e.g. SAMtools, GATK) that use SAM. HISAT2 is distributed under the GPLv3 license, and it runs on the command line under Linux, Mac OS X and Windows.
Download HISAT2 sources and binaries from the Releases sections on the right side.
Binaries are available for Intel architectures (x86_64) running Linux, and Mac OS X.
Building HISAT2 from source requires a GNU-like environment with GCC, GNU Make and other basics. It should be possible to build HISAT2 on most vanilla Linux installations or on a Mac installation with Xcode installed. HISAT2 can also be built on Windows using Cygwin or MinGW (MinGW recommended). For a MinGW build the choice of what compiler is to be used is important since this will determine if a 32 or 64 bit code can be successfully compiled using it. If there is a need to generate both 32 and 64 bit on the same machine then a multilib MinGW has to be properly installed. MSYS, the zlib library, and depending on architecture pthreads library are also required. We are recommending a 64 bit build since it has some clear advantages in real life research problems. In order to simplify the MinGW setup it might be worth investigating popular MinGW personal builds since these are coming already prepared with most of the toolchains needed.
First, download the source package from the Releases section on the right side.
Unzip the file, change to the unzipped directory, and build the
HISAT2 tools by running GNU make (usually with the command make, but
sometimes with gmake) with no arguments. If building with MinGW, run make
from the MSYS environment.
HISAT2 is using the multithreading software model in order to speed up execution times on SMP architectures where this is possible. On POSIX platforms (like linux, Mac OS, etc) it needs the pthread library. Although it is possible to use pthread library on non-POSIX platform like Windows, due to performance reasons HISAT2 will try to use Windows native multithreading if possible.
For the support of SRA data access in HISAT2, please download and install the NCBI-NGS toolkit.
When running make, specify additional variables as follow.
make USE_SRA=1 NCBI_NGS_DIR=/path/to/NCBI-NGS-directory NCBI_VDB_DIR=/path/to/NCBI-NGS-directory,
where NCBI_NGS_DIR and NCBI_VDB_DIR will be used in Makefile for -I and -L compilation options.
For example,
By adding your new HISAT2 directory to your PATH environment variable, you
ensure that whenever you run hisat2, hisat2-build or hisat2-inspect
from the command line, you will get the version you just installed without
having to specify the entire path. This is recommended for most users. To do
this, follow your operating system's instructions for adding the directory to
your PATH.
If you would like to install HISAT2 by copying the HISAT2 executable files
to an existing directory in your PATH, make sure that you copy all the
executables, including hisat2, hisat2-align-s, hisat2-align-l, hisat2-build, hisat2-build-s, hisat2-build-l, hisat2-inspect, hisat2-inspect-s and
hisat2-inspect-l.
The reporting mode governs how many alignments HISAT2 looks for, and how to report them.
In general, when we say that a read has an alignment, we mean that it has a valid alignment. When we say that a read has multiple alignments, we mean that it has multiple alignments that are valid and distinct from one another.
By default, HISAT2 may soft-clip reads near their 5' and 3' ends. Users can control this behavior by setting different penalties for soft-clipping (--sp) or by disallowing soft-clipping ([--no-softclip]).
Two alignments for the same individual read are "distinct" if they map the same read to different places. Specifically, we say that two alignments are distinct if there are no alignment positions where a particular read offset is aligned opposite a particular reference offset in both alignments with the same orientation. E.g. if the first alignment is in the forward orientation and aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, and the second alignment is also in the forward orientation and also aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, they are not distinct alignments.
Two alignments for the same pair are distinct if either the mate 1s in the two paired-end alignments are distinct or the mate 2s in the two alignments are distinct or both.
HISAT2 searches for up to N distinct, primary alignments for
each read, where N equals the integer specified with the -k parameter.
Primary alignments mean alignments whose alignment score is equal or higher than any other alignments.
It is possible that multiple distinct alignments have the same score.
That is, if -k 2 is specified, HISAT2 will search for at most 2 distinct
alignments. The alignment score for a paired-end alignment equals the sum of the
alignment scores of the individual mates. Each reported read or pair alignment
beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS
field. See the SAM specification for details.
HISAT2 does not "find" alignments in any specific order, so for reads that have more than N distinct, valid alignments, HISAT2 does not guarantee that the N alignments reported are the best possible in terms of alignment score. Still, this mode can be effective and fast in situations where the user cares more about whether a read aligns (or aligns a certain number of times) than where exactly it originated.
When HISAT2 finishes running, it prints messages summarizing what happened. These messages are printed to the "standard error" ("stderr") filehandle. For datasets consisting of unpaired reads, the summary might look like this:
20000 reads; of these:
20000 (100.00%) were unpaired; of these:
1247 (6.24%) aligned 0 times
18739 (93.69%) aligned exactly 1 time
14 (0.07%) aligned >1 times
93.77% overall alignment rate
For datasets consisting of pairs, the summary might look like this:
10000 reads; of these:
10000 (100.00%) were paired; of these:
650 (6.50%) aligned concordantly 0 times
8823 (88.23%) aligned concordantly exactly 1 time
527 (5.27%) aligned concordantly >1 times
----
650 pairs aligned concordantly 0 times; of these:
34 (5.23%) aligned discordantly 1 time
----
616 pairs aligned 0 times concordantly or discordantly; of these:
1232 mates make up the pairs; of these:
660 (53.57%) aligned 0 times
571 (46.35%) aligned exactly 1 time
1 (0.08%) aligned >1 times
96.70% overall alignment rate
The indentation indicates how subtotals relate to totals.
The hisat2, hisat2-build and hisat2-inspect executables are actually
wrapper scripts that call binary programs as appropriate. The wrappers shield
users from having to distinguish between "small" and "large" index formats,
discussed briefly in the following section. Also, the hisat2 wrapper
provides some key functionality, like the ability to handle compressed inputs,
and the functionality for --un, --al and related options.
It is recommended that you always run the hisat2 wrappers and not run the binaries directly.
hisat2-build can index reference genomes of any size. For genomes less than
about 4 billion nucleotides in length, hisat2-build builds a "small" index
using 32-bit numbers in various parts of the index. When the genome is longer,
hisat2-build builds a "large" index using 64-bit numbers. Small indexes are
stored in files with the .ht2 extension, and large indexes are stored in
files with the .ht2l extension. The user need not worry about whether a
particular index is small or large; the wrapper scripts will automatically build
and use the appropriate index.
-
If your computer has multiple processors/cores, use
-pThe
-poption causes HISAT2 to launch a specified number of parallel search threads. Each thread runs on a different processor/core and all threads find alignments in parallel, increasing alignment throughput by approximately a multiple of the number of threads (though in practice, speedup is somewhat worse than linear).
Some HISAT2 options specify a function rather than an individual number or
setting. In these cases the user specifies three parameters: (a) a function
type F, (b) a constant term B, and (c) a coefficient A. The available
function types are constant (C), linear (L), square-root (S), and natural
log (G). The parameters are specified as F,B,A - that is, the function type,
the constant term, and the coefficient are separated by commas with no
whitespace. The constant term and coefficient may be negative and/or
floating-point numbers.
For example, if the function specification is L,-0.4,-0.6, then the function
defined is:
f(x) = -0.4 + -0.6 * x
If the function specification is G,1,5.4, then the function defined is:
f(x) = 1.0 + 5.4 * ln(x)
See the documentation for the option in question to learn what the parameter x
is for. For example, in the case if the --score-min option, the function
f(x) sets the minimum alignment score necessary for an alignment to be
considered valid, and x is the read length.
hisat2 [options]* -x <hisat2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <hit>]
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The basename of the index for the reference genome. The basename is the name of
any of the index files up to but not including the final |
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Comma-separated list of files containing mate 1s (filename usually includes
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Comma-separated list of files containing mate 2s (filename usually includes
|
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Comma-separated list of files containing unpaired reads to be aligned, e.g.
|
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Comma-separated list of SRA accession numbers, e.g. |
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File to write SAM alignments to. By default, alignments are written to the "standard out" or "stdout" filehandle (i.e. the console). |
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Reads (specified with |
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Reads (specified with |
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Reads (specified with |
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Reads (specified with |
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The read sequences are given on command line. I.e. |
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Skip (i.e. do not align) the first |
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Align the first |
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Trim |
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Trim |
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Input qualities are ASCII chars equal to the Phred quality plus 33. This is also called the "Phred+33" encoding, which is used by the very latest Illumina pipelines. |
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Input qualities are ASCII chars equal to the Phred quality plus 64. This is also called the "Phred+64" encoding. |
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Convert input qualities from Solexa (which can be negative) to Phred (which can't). This scheme was used in older Illumina GA Pipeline versions (prior to 1.3). Default: off. |
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Quality values are represented in the read input file as space-separated ASCII
integers, e.g., |
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Sets a function governing the maximum number of ambiguous characters (usually
|
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When calculating a mismatch penalty, always consider the quality value at the
mismatched position to be the highest possible, regardless of the actual value.
I.e. input is treated as though all quality values are high. This is also the
default behavior when the input doesn't specify quality values (e.g. in |
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If |
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Sets the maximum ( |
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Sets the maximum ( |
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Disallow soft-clipping. |
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Sets penalty for positions where the read, reference, or both, contain an
ambiguous character such as |
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Sets the read gap open ( |
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Sets the reference gap open ( |
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Sets a function governing the minimum alignment score needed for an alignment to
be considered "valid" (i.e. good enough to report). This is a function of read
length. For instance, specifying |
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Sets the penalty for each pair of canonical splice sites (e.g. GT/AG). Default: 0. |
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Sets the penalty for each pair of non-canonical splice sites (e.g. non-GT/AG). Default: 12. |
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Sets the penalty for long introns with canonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1 |
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Sets the penalty for long introns with noncanonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1 |
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Sets minimum intron length. Default: 20 |
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Sets maximum intron length. Default: 500000 |
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With this mode, you can provide a list of known splice sites, which HISAT2 makes use of to align reads with small anchors. |
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In this mode, HISAT2 reports a list of splice sites in the file : |
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With this mode, you can provide a list of novel splice sites that were generated from the above option "--novel-splicesite-outfile". |
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HISAT2, by default, makes use of splice sites found by earlier reads to align later reads in the same run,
in particular, reads with small anchors (<= 15 bp). |
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Disable spliced alignment. |
[`--rna-strandness`]: #hisat2-options-rna-strandness
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Specify strand-specific information: the default is unstranded. (TopHat has a similar option, --library-type option, where fr-firststrand corresponds to R and RF; fr-secondstrand corresponds to F and FR.) |
[`--tmo/--transcriptome-mapping-only`]: #hisat2-options-tmo
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Report only those alignments within known transcripts. |
[`--dta/--downstream-transcriptome-assembly`]: #hisat2-options-dta
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Report alignments tailored for transcript assemblers including StringTie. With this option, HISAT2 requires longer anchor lengths for de novo discovery of splice sites. This leads to fewer alignments with short-anchors, which helps transcript assemblers improve significantly in computation and memory usage. |
[`--dta-cufflinks`]: #hisat2-options-dta-cufflinks
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Report alignments tailored specifically for Cufflinks. In addition to what HISAT2 does with the above option (--dta), With this option, HISAT2 looks for novel splice sites with three signals (GT/AG, GC/AG, AT/AC), but all user-provided splice sites are used irrespective of their signals. HISAT2 produces an optional field, XS:A:[+-], for every spliced alignment. |
[`--avoid-pseudogene`]: #hisat2-options-avoid-pseudogene
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Try to avoid aligning reads to pseudogenes. Note this option is experimental and needs further investigation. |
[`--no-templatelen-adjustment`]: #hisat2-options-no-templatelen-adjustment
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Disables template length adjustment for RNA-seq reads. |
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It searches for at most Note: HISAT2 is not designed with large values for |
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HISAT2, like other aligners, uses seed-and-extend approaches. HISAT2 tries to extend seeds to full-length alignments. In HISAT2, --max-seeds is used to control the maximum number of seeds that will be extended. HISAT2 extends up to these many seeds and skips the rest of the seeds. Large values for |
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Report secondary alignments. |
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The minimum fragment length for valid paired-end alignments.This option is valid only with --no-spliced-alignment.
E.g. if The larger the difference between Default: 0 (essentially imposing no minimum) |
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The maximum fragment length for valid paired-end alignments. This option is valid only with --no-spliced-alignment.
E.g. if The larger the difference between Default: 500. |
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The upstream/downstream mate orientations for a valid paired-end alignment
against the forward reference strand. E.g., if |
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By default, when |
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By default, |
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Print the wall-clock time required to load the index files and align the reads. This is printed to the "standard error" ("stderr") filehandle. Default: off. |
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Write unpaired reads that fail to align to file at |
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Write unpaired reads that align at least once to file at |
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Write paired-end reads that fail to align concordantly to file(s) at |
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Write paired-end reads that align concordantly at least once to file(s) at
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Print nothing besides alignments and serious errors. |
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Print alignment summary to this file. |
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Print alignment summary in a new style, which is more machine-friendly. |
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Write |
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Write |
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Write a new |
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Suppress SAM records for reads that failed to align. |
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Suppress SAM header lines (starting with |
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Suppress |
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Set the read group ID to |
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Add |
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Remove 'chr' from reference names in alignment (e.g., chr18 to 18) |
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Add 'chr' to reference names in alignment (e.g., 18 to chr18) |
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When printing secondary alignments, HISAT2 by default will write out the |
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Override the offrate of the index with |
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Launch |
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Guarantees that output SAM records are printed in an order corresponding to the
order of the reads in the original input file, even when |
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Use memory-mapped I/O to load the index, rather than typical file I/O.
Memory-mapping allows many concurrent |
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Filter out reads for which the QSEQ filter field is non-zero. Only has an
effect when read format is |
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Use |
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Normally, HISAT2 re-initializes its pseudo-random generator for each read. It
seeds the generator with a number derived from (a) the read name, (b) the
nucleotide sequence, (c) the quality sequence, (d) the value of the |
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Print version information and quit. |
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Print usage information and quit. |
Following is a brief description of the SAM format as output by hisat2.
For more details, see the SAM format specification.
By default, hisat2 prints a SAM header with @HD, @SQ and @PG lines.
When one or more --rg arguments are specified, hisat2 will also print
an @RG line that includes all user-specified --rg tokens separated by
tabs.
Each subsequent line describes an alignment or, if the read failed to align, a read. Each line is a collection of at least 12 fields separated by tabs; from left to right, the fields are:
-
Name of read that aligned.
Note that the SAM specification disallows whitespace in the read name. If the read name contains any whitespace characters, HISAT2 will truncate the name at the first whitespace character. This is similar to the behavior of other tools.
-
Sum of all applicable flags. Flags relevant to HISAT2 are:
1The read is one of a pair
2The alignment is one end of a proper paired-end alignment
4The read has no reported alignments
8The read is one of a pair and has no reported alignments
16The alignment is to the reverse reference strand
32The other mate in the paired-end alignment is aligned to the reverse reference strand
64The read is mate 1 in a pair
128The read is mate 2 in a pair
Thus, an unpaired read that aligns to the reverse reference strand will have flag 16. A paired-end read that aligns and is the first mate in the pair will have flag 83 (= 64 + 16 + 2 + 1).
-
Name of reference sequence where alignment occurs
-
1-based offset into the forward reference strand where leftmost character of the alignment occurs
-
Mapping quality. Mapping quality of HISAT2
-
CIGAR string representation of alignment
-
Name of reference sequence where mate's alignment occurs. Set to
=if the mate's reference sequence is the same as this alignment's, or*if there is no mate. -
1-based offset into the forward reference strand where leftmost character of the mate's alignment occurs. Offset is 0 if there is no mate.
-
Inferred fragment length. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if the mates did not align concordantly. However, size is non-0 if the mates aligned discordantly to the same chromosome.
-
Read sequence (reverse-complemented if aligned to the reverse strand)
-
ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the Phred quality scale and the encoding is ASCII-offset by 33 (ASCII char
!), similarly to a FASTQ file. -
Optional fields. Fields are tab-separated.
hisat2outputs zero or more of these optional fields for each alignment, depending on the type of the alignment:AS:i:<N>Alignment score. Can be negative. Only present if SAM record is for an aligned read.
ZS:i:<N>Alignment score for the best-scoring alignment found other than the alignment reported. Can be negative. Only present if the SAM record is for an aligned read and more than one alignment was found for the read. Note that, when the read is part of a concordantly-aligned pair, this score could be greater than [`AS:i`]. YS:i:<N>Alignment score for opposite mate in the paired-end alignment. Only present if the SAM record is for a read that aligned as part of a paired-end alignment.
XN:i:<N>The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read.
XM:i:<N>The number of mismatches in the alignment. Only present if SAM record is for an aligned read.
XO:i:<N>The number of gap opens, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
XG:i:<N>The number of gap extensions, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
NM:i:<N>The edit distance; that is, the minimal number of one-nucleotide edits (substitutions, insertions and deletions) needed to transform the read string into the reference string. Only present if SAM record is for an aligned read.
YF:Z:<S>String indicating reason why the read was filtered out. See also: [Filtering]. Only appears for reads that were filtered out.
YT:Z:<S>Value of
UUindicates the read was not part of a pair. Value ofCPindicates the read was part of a pair and the pair aligned concordantly. Value ofDPindicates the read was part of a pair and the pair aligned discordantly. Value ofUPindicates the read was part of a pair but the pair failed to aligned either concordantly or discordantly.MD:Z:<S>A string representation of the mismatched reference bases in the alignment. See SAM format specification for details. Only present if SAM record is for an aligned read.
XS:A:<A>Values of
+and-indicate the read is mapped to transcripts on sense and anti-sense strands, respectively. Spliced alignments need to have this field, which is required in Cufflinks and StringTie.
We can report this field for the canonical-splice site (GT/AG), but not for non-canonical splice sites. You can direct HISAT2 not to output such alignments (involving non-canonical splice sites) using "--pen-noncansplice 1000000".NH:i:<N>The number of mapped locations for the read or the pair.
Zs:Z:<S>When the alignment of a read involves SNPs that are in the index, this option is used to indicate where exactly the read involves the SNPs. This optional field is similar to the above MD:Z field. For example,
Zs:Z:1|S|rs3747203,97|S|rs16990981indicates the second base of the read corresponds to a known SNP (ID: rs3747203). 97 bases after the third base (the base after the second one), the read at 100th base involves another known SNP (ID: rs16990981). 'S' indicates a single nucleotide polymorphism. 'D' and 'I' indicate a deletion and an insertion, respectively.
hisat2-build builds a HISAT2 index from a set of DNA sequences.
hisat2-build outputs a set of 6 files with suffixes .1.ht2, .2.ht2,
.3.ht2, .4.ht2, .5.ht2, .6.ht2, .7.ht2, and .8.ht2. In the case of a large
index these suffixes will have a ht2l termination. These files together
constitute the index: they are all that is needed to align reads to that
reference. The original sequence FASTA files are no longer used by HISAT2
once the index is built.
Use of Karkkainen's blockwise algorithm allows hisat2-build to trade off
between running time and memory usage. hisat2-build has three options
governing how it makes this trade: [-p/--packed], --bmax/--bmaxdivn,
and --dcv. By default, hisat2-build will automatically search for the
settings that yield the best running time without exhausting memory. This
behavior can be disabled using the -a/--noauto option.
The indexer provides options pertaining to the "shape" of the index, e.g.
--offrate governs the fraction of Burrows-Wheeler
rows that are "marked" (i.e., the density of the suffix-array sample; see the
original FM Index paper for details). All of these options are potentially
profitable trade-offs depending on the application. They have been set to
defaults that are reasonable for most cases according to our experiments. See
Performance tuning for details.
hisat2-build can generate either small or large indexes. The wrapper
will decide which based on the length of the input genome. If the reference
does not exceed 4 billion characters but a large index is preferred, the user
can specify --large-index to force hisat2-build to build a large index
instead.
The HISAT2 index is based on the FM Index of Ferragina and Manzini, which in turn is based on the Burrows-Wheeler transform. The algorithm used to build the index is based on the blockwise algorithm of Karkkainen.
Usage:
hisat2-build [options]* <reference_in> <ht2_base>
If you use --snp, --ss, and/or --exon, hisat2-build will need about 200GB RAM for the human genome size as index building involves a graph construction.
Otherwise, you will be able to build an index on your desktop with 8GB RAM.
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A comma-separated list of FASTA files containing the reference sequences to be
aligned to, or, if |
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The basename of the index files to write. By default, |
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The reference input files (specified as |
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The reference sequences are given on the command line. I.e. |
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Force |
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Disable the default behavior whereby |
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The maximum number of suffixes allowed in a block. Allowing more suffixes per
block makes indexing faster, but increases peak memory usage. Setting this
option overrides any previous setting for |
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The maximum number of suffixes allowed in a block, expressed as a fraction of
the length of the reference. Setting this option overrides any previous setting
for |
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Use |
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Disable use of the difference-cover sample. Suffix sorting becomes quadratic-time in the worst case (where the worst case is an extremely repetitive reference). Default: off. |
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Do not build the |
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Build only the |
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To map alignments back to positions on the reference sequences, it's necessary
to annotate ("mark") some or all of the Burrows-Wheeler rows with their
corresponding location on the genome.
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The ftab is the lookup table used to calculate an initial Burrows-Wheeler
range with respect to the first |
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This option governs how many rows get marked in a local index:
the indexer will mark every 2^ |
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The local ftab is the lookup table in a local index. The default setting is 6 (ftab is 8KB per local index). |
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Launch |
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Provide a list of SNPs (in the HISAT2's own format) as follows (five columns). SNP ID For example, rs58784443 single 13 18447947 T Use |
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Provide a list of haplotypes (in the HISAT2's own format) as follows (five columns). Haplotype ID For example, ht35 13 18446877 18446945 rs12381094,rs12381056,rs192016659,rs538569910 See the above option, --snp, about how to extract haplotypes. This option is not required, but haplotype information can keep the index construction from exploding and reduce the index size substantially. |
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Note this option should be used with the following --exon option. Provide a list of splice sites (in the HISAT2's own format) as follows (four columns). chromosome name Use |
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Note this option should be used with the above --ss option. Provide a list of exons (in the HISAT2's own format) as follows (three columns). chromosome name Use |
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Use |
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Index only the first |
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Print usage information and quit. |
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Print version information and quit. |
hisat2-inspect extracts information from a HISAT2 index about what kind of
index it is and what reference sequences were used to build it. When run without
any options, the tool will output a FASTA file containing the sequences of the
original references (with all non-A/C/G/T characters converted to Ns).
It can also be used to extract just the reference sequence names using the
-n/--names option or a more verbose summary using the -s/--summary
option.
Usage:
hisat2-inspect [options]* <ht2_base>
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The basename of the index to be inspected. The basename is name of any of the
index files but with the |
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When printing FASTA output, output a newline character every |
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Print reference sequence names, one per line, and quit. |
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Print a summary that includes information about index settings, as well as the names and lengths of the input sequences. The summary has this format: Fields are separated by tabs. Colorspace is always set to 0 for HISAT2. |
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Print SNPs, and quit. |
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Print splice sites, and quit. |
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Print splice sites including those not in the global index, and quit. |
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Print exons, and quit. |
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Print verbose output (for debugging). |
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Print version information and quit. |
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Print usage information and quit. |
HISAT2 comes with some example files to get you started. The example files are not scientifically significant; these files will simply let you start running HISAT2 and downstream tools right away.
First follow the manual instructions to obtain HISAT2. Set the HISAT2_HOME
environment variable to point to the new HISAT2 directory containing the
hisat2, hisat2-build and hisat2-inspect binaries. This is important,
as the HISAT2_HOME variable is used in the commands below to refer to that
directory.
To create an index for the genomic region (1 million bps from the human chromosome 22 between 20,000,000 and 20,999,999) included with HISAT2, create a new temporary directory (it doesn't matter where), change into that directory, and run:
$HISAT2_HOME/hisat2-build $HISAT2_HOME/example/reference/22_20-21M.fa --snp $HISAT2_HOME/example/reference/22_20-21M.snp 22_20-21M_snp
The command should print many lines of output then quit. When the command
completes, the current directory will contain ten new files that all start with
22_20-21M_snp and end with .1.ht2, .2.ht2, .3.ht2, .4.ht2, .5.ht2, .6.ht2,
.7.ht2, and .8.ht2. These files constitute the index - you're done!
You can use hisat2-build to create an index for a set of FASTA files obtained
from any source, including sites such as UCSC, NCBI, and Ensembl. When
indexing multiple FASTA files, specify all the files using commas to separate
file names. For more details on how to create an index with hisat2-build,
see the manual section on index building. You may also want to bypass this
process by obtaining a pre-built index.
Stay in the directory created in the previous step, which now contains the
22_20-21M index files. Next, run:
$HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -U $HISAT2_HOME/example/reads/reads_1.fa -S eg1.sam
This runs the HISAT2 aligner, which aligns a set of unpaired reads to the
genome region using the index generated in the previous step.
The alignment results in SAM format are written to the file eg1.sam, and a
short alignment summary is written to the console. (Actually, the summary is
written to the "standard error" or "stderr" filehandle, which is typically
printed to the console.)
To see the first few lines of the SAM output, run:
head eg1.sam
You will see something like this:
@HD VN:1.0 SO:unsorted
@SQ SN:22:20000001-21000000 LN:1000000
@PG ID:hisat2 PN:hisat2 VN:2.0.0-beta
1 0 22:20000001-21000000 397984 255 100M * 0 0 GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU NH:i:1
2 16 22:20000001-21000000 398131 255 100M * 0 0 ATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:80A19 YT:Z:UU NH:i:1 Zs:Z:80|S|rs576159895
3 16 22:20000001-21000000 398222 255 100M * 0 0 TGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:16A83 YT:Z:UU NH:i:1 Zs:Z:16|S|rs2629364
4 16 22:20000001-21000000 398247 255 90M200N10M * 0 0 CAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAACCGCTGCACCAGGAAGTCGATCCAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU XS:A:- NH:i:1
5 16 22:20000001-21000000 398194 255 100M * 0 0 GGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:17A26A55 YT:Z:UU NH:i:1 Zs:Z:17|S|rs576159895,26|S|rs2629364
6 0 22:20000001-21000000 398069 255 100M * 0 0 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU NH:i:1
7 0 22:20000001-21000000 397896 255 100M * 0 0 GTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:31G68 YT:Z:UU NH:i:1 Zs:Z:31|S|rs562662261
8 0 22:20000001-21000000 398150 255 100M * 0 0 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:61A26A11 YT:Z:UU NH:i:1 Zs:Z:61|S|rs576159895,26|S|rs2629364
9 16 22:20000001-21000000 398329 255 8M200N92M * 0 0 ACCAGGAAGTCGATCCAGATGTAGTGGGGGGTCACTTCGGGGGGACAGGGTTTGGGTTGACTTGCTTCCGAGGCAGCCAGGGGGTCTGCTTCCTTTATCT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU XS:A:- NH:i:1
10 16 22:20000001-21000000 398184 255 100M * 0 0 CTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:27A26A45 YT:Z:UU NH:i:1 Zs:Z:27|S|rs576159895,26|S|rs2629364
The first few lines (beginning with @) are SAM header lines, and the rest of
the lines are SAM alignments, one line per read or mate. See the HISAT2
manual section on SAM output and the SAM specification for details about how
to interpret the SAM file format.
To align paired-end reads included with HISAT2, stay in the same directory and run:
$HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam
This aligns a set of paired-end reads to the reference genome, with results
written to the file eg2.sam.
SAMtools is a collection of tools for manipulating and analyzing SAM and BAM
alignment files. BCFtools is a collection of tools for calling variants and
manipulating VCF and BCF files, and it is typically distributed with SAMtools.
Using these tools together allows you to get from alignments in SAM format to
variant calls in VCF format. This example assumes that samtools and
bcftools are installed and that the directories containing these binaries are
in your PATH environment variable.
Run the paired-end example:
$HISAT2_HOME/hisat -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam
Use samtools view to convert the SAM file into a BAM file. BAM is a the
binary format corresponding to the SAM text format. Run:
samtools view -bS eg2.sam > eg2.bam
Use samtools sort to convert the BAM file to a sorted BAM file. The following command requires samtools version 1.2 or higher.
samtools sort eg2.bam -o eg2.sorted.bam
We now have a sorted BAM file called eg2.sorted.bam. Sorted BAM is a useful
format because the alignments are (a) compressed, which is convenient for
long-term storage, and (b) sorted, which is convenient for variant discovery.
To generate variant calls in VCF format, run:
samtools mpileup -uf $HISAT2_HOME/example/reference/22_20-21M.fa eg2.sorted.bam | bcftools view -bvcg - > eg2.raw.bcf
Then to view the variants, run:
bcftools view eg2.raw.bcf
See the official SAMtools guide to Calling SNPs/INDELs with SAMtools/BCFtools for more details and variations on this process.