File Formats

QSeq file format

The QSeq file format is fully documented in the Illumina pipeline user’s guide (page 163). In brief, the file format is as follows.

• One record per line
• Each record represents one read.
• Unknown sequence bases are represented as ‘.’

Each record includes the following tab-separated fields, in order.

Machine name

(hopefully) Unique identifier of the sequencer.

Run number

(hopefully) Unique number to identify the run on the sequencer.

Lane number

Positive integer (currently 1-8).

Tile number

Positive integer.

X

X coordinate of the spot. Integer (can be negative).

Y

Y coordinate of the spot. Integer (can be negative).

Index

Sample tag in multiplexed runs. For regular runs without multiplexing this field should have a value of 0.

Read Number

1 for single reads; 1 or 2 for paired ends.

Sequence

The base sequence for this read. Unknown bases are indicated with a ‘.’

Quality

The calibrated base quality string.

Filter

Did the read pass filtering? 0 - No, 1 - Yes.

Example

Here are two sample lines from a QSeq file. Order doesn’t matter, and read mates do not have to be in the same file or any particular relative position

CRESSIA       242     1       2204    1453    1918    0       1       .TTAATAAGAATGTCTGTTGTGGCTTAAAA  B[[[W][Y[Zccccccccc\cccac_____  1
CRESSIA       242     1       2204    1490    1921    0       2       ..GTAAAACCCATATATTGAAAACTACAAA  BWUTWcXVXXcccc_cccccccccc_cccc  1

PRQ file format

The PRQ file format is another line-oriented format. Each record contains a read pair in the following tab-separated fields.

Id

A unique id for the read pair.

Read 1 sequence

The base sequence for read 1. Unknown bases are indicated with a ‘N’

Quality 1

The base quality sequence for read 1 in Sanger Phred+33 encoding.

Read 2 sequence

The base sequence for read 2. Unknown bases are indicated with a ‘N’

Quality 2

The base quality sequence for read 2 in Sanger Phred+33 encoding.

Example

Here is a sample line from a PRQ file, constructed with the QSeq lines above:

CRESSIA_242:1:2204:1453;1918#0        NTTAATAAGAATGTCTGTTGTGGCTTAAAA  #<<<8><:<;DDDDDDDDD=DDDBD@@@@@  NNGTAAAACCCATATATTGAAAACTACAAA  #8658D9799DDDD@DDDDDDDDDD@DDDD

Fastq file format

The Fastq is another text-based file format, quite popular although perhaps only for historical reasons.

As of version 1.8 of Illumina’s Casava software, Illumina is returning to the fastq format (from the qseq format). For this reason we have implemented fastq input in Seal PairReadsQseq.

Seal by default assumes the Illumina-style fastq format (see the Casava v. 1.8 user’s guide p. 41). This Fastq format is defined as a series of records. Each record consists of 4 lines:

1. begins with ‘@’ and followed by the sequence identifier and meta info;
2. the raw sequence;
3. begins with ‘+’ and is optionally followed by the same sequence identifier as in line 1;
4. ASCII base quality values in the Sanger-style Phred+33 encoding.

In Illumina Fastq files the identifier line (line 1) contains several fields of meta info about the sequence in the following format:

@<Instrument>:<Run Number>:<Flowcell ID>:<Lane>:<Tile>:<X-pos>:<Y-pos>SPACE<Read>:<Is Filtered>:<Control Number>:<Index Sequence>

The meaning of each field is as follows.

Instrument

(hopefully) Unique identifier of the sequencer.

Run Number

Run number on the sequencer.

Flowcell ID

The id of the flowcell used in the run that produced the read.

Lane

Flowcell lane number. Positive integer (currently 1-8).

Tile number

Tile number within the lane. Positive integer.

X-pos

X coordinate of the cluster within the tile. Integer (can be negative).

Y-pos

Y coordinate of the cluster within the tile. Integer (can be negative).

Read

Read number. 1 for single reads; 1 or 2 for paired ends.

Is Filtered

Did the read fail filtering? Y or N.

Control Number

0 when none of the control bits are on, otherwise it is an even number

Index Sequence

Sample tag in multiplexed runs.

The Y-pos and Read fields are separated by a SPACE character, while the rest of the fields are separated by colon characters.

Example

Here is an example of a fastq record from the Casava documentation:

@EAS139:136:FC706VJ:2:5:1000:12850  1:Y:18:ATCACG
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
+
BBBBCCCC?<A?BC?7@@???????DBBA@@@@A@@

Standard Fastq files

The format specified above for the id line of the fastq files has been invented by Illumina and, as far as we know, is only used by Casava. The “standard” fastq format makes no specifications for the id line and gives no means to express meta information about the read. Still, Seal tries to let you work with “plain” fastq files, as long as their id ends with a “/1” or “/2” so that it can extract the read number for the sequence.

Seal will initially try to read a Fastq file as an Illumina file, and then revert to the standard format after the first record that doesn’t match the Illumina format.